Scanning Analyzer for Single Molecule Detection and Methods of Use

ABSTRACT

The invention encompasses analyzers and analyzer systems that include a single molecule analyzer, methods of using the analyzer and analyzer systems to analyze samples, either for single molecules or for molecular complexes. The single molecule uses electromagnetic radiation that is translated through the sample to detect the presence or absence of a single molecule. The single molecule analyzer provided herein is useful for diagnostics because the analyzer detects single molecules with zero carryover between samples.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/015,142, filed Dec. 19, 2007, which application is incorporatedherein by reference.

BACKGROUND

Advances in biomedical research, medical diagnosis, prognosis,monitoring and treatment selection, bioterrorism detection, and otherfields involving the analysis of multiple samples of low volume andconcentration of analytes have led to development of sample analysissystems capable of sensitively detecting particles in a sample atever-decreasing concentrations. U.S. Pat. Nos. 4,793,705 and 5,209,834describe previous systems that achieved extremely sensitive detection.The present invention provides further development in this field.

SUMMARY OF THE INVENTION

Provided herein is a single molecule analyzer comprising: (a) anelectromagnetic radiation source for providing electromagnetic radiationto a sample container that comprises a sample; (b) a system fordirecting the electromagnetic radiation from the electromagneticradiation source to an interrogation space in the sample; (c) atranslating system for translating the interrogation space through atleast a portion of the sample, thereby forming a moveable interrogationspace; and (d) a detector operably connected to the interrogation spaceto detect electromagnetic radiation emitted from a single molecule inthe interrogation space if the molecule is present. In some embodiments,the single molecule analyzer has a translating system wherein thetranslating system is capable of translating the interrogation space inone or more of a linear and a non-linear path. In some embodiments, thenon-linear path is substantially a circular path. In some embodiments,the non-linear path is substantially a helical pattern. In someembodiments, the non-linear path is substantially a raster pattern. Insome embodiments, the single molecule analyzer described herein furthercomprises a container with a surface adapted and configured forcontaining and confining at least one sample on the surface. In someembodiments, the container is a plate. In further embodiments, the plateis a microtiter plate.

In some embodiments of the single molecule analyzer, the interrogationspace has an effective volume of more than about 1 μm³, more than about2 μm³, more than about 3 μm³, more than about 4 μm³, more than about 5μm³, more than about 10 μm³, more than about 15 μm³, more than about 30μm³, more than about 50 μm³, more than about 75 μm³, more than about 100μm³, more than about 150 μm³, more than about 200 μm³, more than about250 μm³, more than about 300 μm³, more than about 400 μm³, more thanabout 450 μm³, more than about 500 μm³, more than about 550 μm³, morethan about 600 μm³, more than about 750 μm³, more than about 1000 μm³,more than about 2000 μm³, more than about 4000 μm³, more than about 6000μm³, more than about 8000 μm³, more than about 10000 μm³, more thanabout 12000 μm³, more than about 13000 μm³, more than about 14000 μm³,more than about 15000 μm³, more than about 20000 μm³, more than about30000 μm³, more than about 40000 μm³, or more than about 50000 μm³. Insome embodiments, the interrogation space is of a volume less than about50000 μm³, less than about 40000 μm³, less than about 30000 μm³, lessthan about 20000 μm³, less than about 15000 μm³, less than about 14000μm³, less than about 13000 μm³, less than about 12000 μm³, less thanabout 11000 μm³, less than about 9500 μm³, less than about 8000 μm³,less than about 6500 μm³, less than about 6000 μm³, less than about 5000μm³, less than about 4000 μm³, less than about 3000 μm³, less than about2500 μm³, less than about 2000 μm³, less than about 1500 μm³, less thanabout 1000 μm³, less than about 800 μm³, less than about 600 μm³, lessthan about 400 μm³, less than about 200 μm³, less than about 100 μm³,less than about 75 μm³, less than about 50 μm³, less than about 25 μm³,less than about 20 μm³, less than about 15 μm³, less than about 14 μm³,less than about 13 μm³, less than about 12 μm³, less than about 11 μm³,less than about 10 μm³, less than about 5 m, less than about 4 μm³, lessthan about 3 μm³, less than about 2 μm³, or less than about 1 μm³. Insome embodiments, the volume of the interrogation space is between about1 μm³ and about 10000 μm³. In some embodiments, the interrogation spaceis between about 1 μm³ and about 1000 μm³. In some embodiments, theinterrogation space is between about 1 μm³ and about 100 μm³. In someembodiments, the interrogation space is between about 1 μm³ and about 50μm³. In some embodiments the interrogation space is between about 1 μm³and about 10 μm³. In some embodiments, the interrogation space isbetween about 2 μm³ and about 10 μm³. In some embodiments, theinterrogation space is between about 3 μm³ and about 7 μm³. In someembodiments, the interrogation space is between about 15 μm³ and about11000 μm³. In some embodiments, the interrogation space is between about200 μm³ and about 3000 μm³. In some embodiments, the interrogation spaceis between about 500 μm³ and about 600 μm³.

In some embodiments of the single molecule analyzer, the singlemolecules are attached to the surface of the container. In someembodiments, the single molecules are attached to the surface of thecontainer by a noncovalent bond. In a further embodiment, thenoncovalent bonds are formed between the molecules and antibodies thatare covalently or non-covalently bound to the surface of the container.In a further embodiment, the noncovalent bonds are formed between themolecules and antibodies located on the surface of the container. Insome embodiments, the single molecule analyzer further comprises amicroscope objective wherein a depth of field of the microscopeobjective and a lateral extent of the laser beam together define theinterrogation space. In some embodiments, the depth of field and adiameter of the aperture imaged to the microscope objective togetherdefine the interrogation space. In some embodiments, the microscopeobjective is adapted and configured to collect the electromagneticradiation emitted from a single molecule located within theinterrogation space. In some embodiments, the interrogation space iscapable of being translated through a portion of a sample. In someembodiments, the translating system is constructed and arranged totranslate through the portion of sample more than one time. In someembodiments, the translating system is constructed and arranged totranslate through a same portion of sample a first time and a secondtime at a sufficiently slow speed as to allow a molecule of interestthat is detected the first time the interrogation space is translatedthrough the portion of sample to substantially diffuse out of theportion of sample after the first time the portion of sample isinterrogated by the interrogation space, and to further allow asubsequent molecule of interest, if present, to substantially diffuseinto the portion of sample the second time the portion of sample isinterrogated by the interrogation space. In some embodiments, thetranslating system is constructed and arranged to translate theinterrogation space such that the detection spot returns to the portionof sample after sufficient time has passed so that molecules detected inthe first pass can diffuse out of the portion, and other molecules candiffuse into the portion. In some embodiments, the single moleculeanalyzer further comprises a system capable of translating theinterrogation space in a substantially circular pattern. In such anembodiment, the system is capable of translating the interrogation spaceat a speed of between about 100 and about 1000 RPM. In some embodiments,the scan speed of the interrogation space is more than 100 RPM. In someembodiments, the scan speed of the interrogation space is more than 300RPM. In some embodiments, the scan speed of the interrogation space ismore than 500 RPM. In some embodiments, the scan speed of theinterrogation space is more than 700 RPM. In some embodiments, the scanspeed of the interrogation space is more than 900 RPM. In someembodiments, the scan speed of the interrogation space is less than 1000RPM. In some embodiments, the scan speed of the interrogation space isless than 800 RPM. In some embodiments, the scan speed of theinterrogation space is less than 600 RPM. In some embodiments, the scanspeed of the interrogation space is less than 400 RPM. In someembodiments, the scan speed of the interrogation space is less than 200RPM. In some embodiments, the scan speed of the interrogation space isbetween about 100 RPM and about 1000 RPM. In some embodiments, the scanspeed of the interrogation space is between about 200 RPM and about 900RPM. In some embodiments, the scan speed of the interrogation space isbetween about 300 RPM and about 800 RPM. In some embodiments, the scanspeed of the interrogation space is between about 400 RPM and about 700RPM. In some embodiments, the scan speed of the interrogation space isbetween about 450 RPM and about 600 RPM. In some embodiments, the scanspeed of the interrogation space is between about 450 RPM and about 550RPM.

In some embodiments, the single molecule analyzer is adapted andconfigured to sequentially detect the presence or absence of a singlemolecule of a particular type in a first sample, and detect the presenceor absence of a single molecule of the type in a second sample, whereinthere is no carryover between the first and the second sample.

Further provided herein is a microtiter plate comprising: (a) a basecomprising a material substantially transparent to light of wavelengthsbetween 550 nm and 800 nm and comprising one or more portions that areof thickness such that an image can be formed on a first side of theportion by a high numerical aperture lens positioned on a second side ofthe portion and wherein no part of the image is formed within the base;and (b) a surface adapted and configured for containing and confining atleast one fluid sample on the surface. In some embodiments, the base istransparent to light of wavelengths between 600 nm and 750 nm. In someembodiments, the base is transparent to light of wavelengths between 630nm and 740 nm. In some embodiments, the base is transparent to light ofwavelengths between 630 nm and 640 nm. In some embodiments, the platesurface comprises a series of microwells. In some embodiments, the platecomprises a material that emits less fluorescence than polystyrene.

Further provided herein is an instrument capable of sequentiallydetecting the presence or absence of a single molecule of a particulartype in a first sample, and detecting the presence or absence of asingle molecule of the type in a second sample, wherein the instrumentis adapted and configured so that there is no carryover between thefirst and the second sample.

Further provided herein is a method of sequentially detecting thepresence or absence of a single molecule of a particular type in a firstsample, and detecting the presence or absence of a single molecule ofthe type in a second sample, wherein there is no carryover between thefirst and the second sample. In some embodiments a single molecule ofinterest is detected in the first sample and the second sample whereinthe first sample and the second sample are contained and confined in anon-disposable apparatus.

Provided herein is a method for detecting the presence or absence of asingle molecule in a sample comprising: (a) directing electromagneticradiation from an electromagnetic radiation source to an interrogationspace in the sample; (b) detecting the presence or absence of a firstsingle molecule in the interrogation space located at a first positionin the sample; (c) translating the interrogation space through thesample to a subsequent position in the sample; (d) detecting thepresence or absence of a subsequent single molecule in the subsequentposition in the sample; and (e) repeating steps (c) and (d) as requiredto detect the presence or absence of a single molecule in more than oneposition of the sample. In some embodiments of this invention, theinterrogation space has an effective volume of more than about 1 μm³,more than about 2 μm³, more than about 3 μm³, more than about 4 μm³,more than about 5 μm³, more than about 10 μm³, more than about 15 μm³,more than about 30 μm³, more than about 50 μm³, more than about 75 μm³,more than about 100 μm³, more than about 150 μm³, more than about 200μm³, more than about 250 μm³, more than about 300 μm³, more than about400 μm³, more than about 450 μm³, more than about 500 μm³, more thanabout 550 μm³, more than about 600 μm³, more than about 750 μm³, morethan about 1000 μm³, more than about 2000 μm³, more than about 4000 μm³,more than about 6000 μm³, more than about 8000 μm³, more than about10000 μm³, more than about 12000 μm³, more than about 13000 μm³, morethan about 14000 μm³, more than about 15000 μm³, more than about 20000μm³, more than about 30000 μm³, more than about 40000 μm³, or more thanabout 50000 μm³. In some embodiments, the volume of the interrogationspace is less than about 50000 μm³, less than about 40000 μm³, less thanabout 30000 μm³, less than about 20000 μm³, less than about 15000 μm³,less than about 14000 μm³, less than about 13000 μm³, less than about12000 μm³, less than about 11000 μm³, less than about 9500 μm³, lessthan about 8000 μm³, less than about 6500 μm³, less than about 6000 μm³,less than about 5000 μm³, less than about 4000 μm³, less than about 3000μm³, less than about 2500 μm³, less than about 2000 μm³, less than about1500 μm³, less than about 1000 μm³, less than about 800 μm³, less thanabout 600 μm³, less than about 400 μm³, less than about 200 μm³, lessthan about 100 μm³, less than about 75 μm³, less than about 50 μm³, lessthan about 25 μm³, less than about 20 μm³, less than about 15 μm³, lessthan about 14 μm³, less than about 13 μm³, less than about 12 μm³, lessthan about 11 μm³, less than about 10 μm³, less than about 5 μm³, lessthan about 4 μm³, less than about 3 μm³, less than about 2 μm³, or lessthan about 1 μm³. In some embodiments, the volume of the interrogationspace is between about 1 μm³ and about 10000 μm³. In some embodiments,the interrogation space is between about 1 μm³ and about 1000 μm³. Insome embodiments, the interrogation space is between about 1 μm³ andabout 100 μm³. In some embodiments the interrogation space is betweenabout 1 μm³ and about 50 μm³. In some embodiments the interrogationspace is between about 1 μm³ and about 10 μm³. In some embodiments, theinterrogation space is between about 2 μm³ and about 10 μm³. In someembodiments, the interrogation space is between about 3 μm³ and about 7μm³. In some embodiments, the interrogation space is between about 15μm³ and about 11000 μm³. In some embodiments, the interrogation space isbetween about 200 μm³ and about 3000 μm³. In some embodiments, theinterrogation space is between about 500 μm³ and about 600 μm³.

In some embodiments of the method, the interrogation space is translatedin a non-linear path. In a further embodiment, the non-linear pathcomprises a substantially circular path. In a further embodiment, thenon-linear path comprises a substantially helical path. In a furtherembodiment, the sample remains substantially stationary relative to theelectromagnetic radiation directed at the interrogation space locatedwithin the sample. In some embodiments, the sample is translated in thex-y axis and the electromagnetic radiation source is kept substantiallystatic. In some embodiments, both the electromagnetic radiation and thesample are translated relative to each other. In some embodiments, theinterrogation space is translated through the first position of samplemore than one time. In some embodiments, the interrogation space istranslated through the first position of sample a subsequent time at asufficiently slow speed as to allow a molecule of interest, if present,detected the first time the interrogation space is translated throughthe position of sample to substantially diffuse out of the position ofsample after the first time the position of sample is interrogated bythe interrogation space and to further allow a subsequent molecule ofinterest, if present, to substantially diffuse into the position ofsample the second time the position of sample is interrogated by theinterrogation space. In some embodiments, the interrogation space istranslated such that the detection spot returns to the first position ofsample after sufficient time has passed so that molecules detected inthe first pass can diffuse out of the position, and other molecules candiffuse into the position. some embodiments, the method furthercomprising the steps of sequentially detecting the presence or absenceof a single molecule of a particular type in the sample, then detectingthe presence or absence of a single molecule of the same type in asecond sample, wherein there is no carryover between the first and thesecond sample. In some embodiments of the method, the first sample andthe second sample are contained and confined in a non-disposableapparatus.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A illustrates the scanning single molecule analyzer as viewed fromthe top;

FIG. 1B illustrates the scanning single molecule analyzer as viewed fromthe side; and

FIG. 2 depicts a graph showing the diffusion time for a 155 KDamolecular weight molecule as a function of the diffusion radius of themolecule.

FIG. 3 shows detection event data generated using a scanning singlemolecule analyzer.

FIG. 4 shows a standard curve generated with a scanning single moleculeanalyzer by detecting a sample over a range of known concentrations.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The invention provides instruments, kits, compositions, and methods forthe highly sensitive detection of single molecules, and for thedetermination of the concentration of the molecules in a sample. In someembodiments, the sensitivity and precision of the instruments,compositions, methods, and kits of the invention can be achieved by acombination of factors selected from, but not limited to,electromagnetic sources of appropriate wavelength and power output,appropriate interrogation space size, high numerical aperture lenses,detectors capable of detecting single photons, and data analysis systemsfor counting single molecules. The instruments of the invention arereferred to as “single molecule detectors” or “single particledetectors,” and are also encompassed by the terms “single moleculeanalyzers” and “single particle analyzers.” The sensitivity andprecision of the kits and methods of the invention are achieved in someembodiments by the use of the instruments of the invention together witha combination of factors selected from, but not limited to, labels formolecules that exhibit characteristics that allow the molecules to bedetected at the level of the single molecule, and methods assaying thelabel in the instruments described herein.

The instruments, kits, and methods of the invention are especiallyuseful in the sensitive and precise detection of single molecules orsmall molecules, and for the determination of the concentration of themolecules in a sample.

The invention provides, in some embodiments, instruments and kits forthe sensitive detection and determination of concentration of moleculesby detection of single molecules, labels for such detection anddetermination, and methods using such instruments and labels in theanalysis of samples. In particular, the sensitivity and precision of theinstruments, kits, and methods of the invention make possible thedetection and determination of concentration of molecules, e.g., markersfor biological states, at extremely low concentrations, e.g.,concentrations below about 100, 10, 1, 0.1, 0.01, or 0.001 femtomolar.In further embodiments, the instruments and kits of the invention arecapable of determining a concentration of a species in a sample, e.g.,the concentration of a molecule, over a large dynamic range ofconcentrations without the need for dilution or other treatment ofsamples, e.g., over a concentration range of more than 10⁵-fold,10⁶-fold, or 10⁷-fold.

The high sensitivity of the instruments, kits, and methods of theinvention allows the use of markers, e.g., biological markers, whichwere not previously useful because of a lack of sensitivity ofdetection. The high sensitivity of the instruments, kits, and methods ofthe invention also facilitate the establishment of new markers. Thereare numerous markers currently available which could be useful indetermining biological states, but are not currently of practical usebecause of current limitations in measuring their lower concentrationranges. In some cases, abnormally high levels of the marker aredetectable by current methods, but normal ranges are unknown. In somecases, abnormally high levels of the marker are detectable by currentmethods, but normal ranges have not been established. In some cases,upper normal ranges of the marker are detectable, but not lower normalranges, or levels below normal. In some cases, e.g., markers of canceror infection, any level of the marker can indicate the presence of abiological state, and enhancing sensitivity of detection is an advantagefor early diagnosis. In some cases, the rate of change, or lack ofchange, in the concentration of a marker over multiple time pointsprovides the most useful information, but present methods of analysis donot permit time point sampling in the early stages of a condition whenit is typically most treatable. In some cases, the marker can bedetected at clinically useful levels only through the use of cumbersomemethods that are not practical or useful in a clinical setting, such asmethods that require complex sample treatment and time-consuminganalysis. In addition, there are potential markers of biological stateswith sufficiently low concentration that their presence remainsextremely difficult or impossible to detect by current methods.

The analytical methods and compositions of the present invention providelevels of sensitivity, precision, and robustness that allow thedetection of markers for biological states at concentrations at whichthe markers have been previously undetectable, thus allowing the“repurposing” of such markers from confirmatory markers, or markersuseful only in limited research settings, to diagnostic, prognostic,treatment-directing, or other types of markers useful in clinicalsettings and/or in large scale clinical settings, including clinicaltrials. Such methods allow the determination of normal and abnormalranges for such markers.

The markers thus repurposed can be used for, e.g., detection of normalstate (normal ranges), detection of responder/non-responder (e.g., to atreatment, such as administration of a drug); detection of early diseaseor pathological occurrence (e.g., early detection of cancer, earlydetection of cardiac ischemia); disease staging (e.g., cancer); diseasemonitoring (e.g., diabetes monitoring, monitoring for cancer recurrenceafter treatment); study of disease mechanism; and study of treatmenttoxicity, such as toxicity of drug treatments.

The invention thus provides methods and compositions for the sensitivedetection of markers, and further methods of establishing values fornormal and abnormal levels of markers. In further embodiments, theinvention provides methods of diagnosis, prognosis, and/or treatmentselection based on values established for the markers. The inventionalso provides compositions for use in such methods, e.g., detectionreagents for the ultrasensitive detection of markers.

II. Instruments and System for Scanning Analyzer System

The methods of the invention utilize scanning analyzers, e.g., singlemolecule detectors. Such single molecule detectors include embodimentsas hereinafter described.

A. Apparatus/System

In one aspect, the system and methods described herein utilize ascanning analyzer system capable of detecting a single molecule in asample. In one embodiment, the scanning analyzer system is capable ofproviding electromagnetic radiation from an electromagnetic radiationsource to a sample located within a sample container. The singlemolecule analyzer includes a system for directing the electromagneticradiation from the electromagnetic radiation source to an interrogationspace in the sample. The single molecule analyzer also includes atranslating system for translating the interrogation space through atleast a portion of the sample, thereby forming a moveable interrogationspace. In some embodiments, the detector of the single molecule analyzeris operably connected to the interrogation space of the single moleculeanalyzer such that it detects radiation emitted from a single moleculein the interrogation space if the molecule is present.

In one aspect, the scanning analyzer system includes an electromagneticradiation source for exciting a single molecule labeled with afluorescent label. In one embodiment, the electromagnetic radiationsource of the analyzer system is a laser. In a further embodiment, theelectromagnetic radiation source is a continuous wave laser.

In a typical embodiment, the electromagnetic radiation source excites afluorescent moiety attached to a label as the interrogation spaceencounters the label. In some embodiments, the fluorescent label moietyincludes one or more fluorescent dye molecules. In some embodiments, thefluorescent label moiety is a quantum dot. Any suitable fluorescentmoiety as described herein can be used as a label.

In a typical embodiment, the scanning analyzer system includes a systemfor directing the electromagnetic radiation to an interrogation space inthe sample. In some embodiments, the concentration of the sample is suchthat the interrogation space is unlikely to contain more than one singlemolecule of interest; e.g., the interrogation space contains zero or onesingle molecule of interest in most cases. The interrogation space canthen be moved through the sample to detect single molecules locatedthroughout the sample. In a typical embodiment, electromagneticradiation from the electromagnetic radiation source excites afluorescent moiety attached to a label as the electromagnetic radiation,and the interrogation space into which the electromagnetic radiation isdirected, is moved through the sample.

Typically, the scanning analyzer system further includes a translatingsystem for translating the interrogation space through at least aportion of the sample, thereby forming a moveable interrogation space.The moveable interrogation space can detect multiple single molecules ofinterest located in different portions of the sample.

The interrogation space passes over the label and subsequently the labelemits a detectable amount of energy when excited by the electromagneticradiation source. In a typical embodiment, the single molecule analyzercontains a detector operably connected to the interrogation space todetect electromagnetic radiation emitted from a single molecule in theinterrogation space. The electromagnetic radiation detector is capableof detecting the energy emitted by the label, e.g., by the fluorescentmoiety of the label.

B. Scanning Single Molecule Analyzer

As shown in FIGS. 1A and 1B, described herein is one embodiment of ascanning analyzer system 100. The analyzer system 100 includeselectromagnetic radiation source 110, a first alignment mirror 112, asecond alignment mirror 114, a dichroic mirror 160, a rotating scanmirror 122 mounted to the shaft 124 of a scan motor 120. As shown inFIG. 1B, the rotating scan mirror 122 deflects the electromagneticradiation source through a first scan lens 130, through a second scanlens 132, and through a microscope objective lens 140, to a sample plate170. The fluorescence associated with the single molecules located onthe sample plate 170 is detected using a tube lens 180, an aperture 182,a detector filter 188, a detector lens 186, and a detector 184. Thesignal is then processed by a processor (not shown) operatively coupledto the detector 184. In some embodiments, the entire scanning analyzersystem 100 is mounted to a baseboard 190.

In operation the electromagnetic radiation source 110 is aligned so thatits output 126, e.g., a beam, is reflected off the front surface 111 ofa first alignment mirror 112 to the front surface 113 of a secondalignment mirror 114 to the dichroic mirror 160 mounted to a dichroicmirror mount 162. The dichroic mirror 160 then reflects theelectromagnetic radiation 126 to the front surface of a scan mirror 122located at the tip of the shaft 124 of the scan motor 120. Theelectromagnetic radiation 126 then passes through a first scan lens 130and a second scan lens 132 to the microscope objective lens 140. Theobjective lens 140 focuses the beam 126 through the base 174 of thesample plate 170 and directs the beam 126 to an interrogation spacelocated on the opposite side of the sample plate 170 from which the beam126 entered. Passing the electromagnetic radiation beam 126 through afirst scan lens 130 and a second scan lens 132 ensures all light to theobjective lens 140 is coupled efficiently. The beam 126 excites thelabel attached to the single molecule of interest located on the sampleplate 170. The label emits radiation that is collected by the objective140. The electromagnetic radiation is then passed back through the scanlenses 130,132 which then ensure coupling efficiency of the radiationfrom the objective 140. The detected radiation is reflected off of thefront surface of the scan mirror 122 to the dichroic mirror 160. Becausethe fluorescent light detected is different than the color of theelectromagnetic radiation source 110, the fluorescent light passing thedichroic mirror 160 passes through a tube lens 180, an aperture 182, adetector filter 188 and detector lens 186 to a detector 184. Thedetector filter 188 minimizes aberrant noise signals due to lightscatter or ambient light while maximizing the signal emitted by theexcited fluorescent moiety bound to the particle. A processor processesthe light signal from the particle according to the methods describedherein.

In a preferred embodiment, the microscope objective 140 has a numericalaperture. As used herein, “high numerical aperture lens” includes a lenswith a numerical aperture of equal to or greater than 0.6. The numericalaperture is a measure of the number of highly diffracted image-forminglight rays captured by the objective. A higher numerical aperture allowsincreasingly oblique rays to enter the objective lens and therebyproduce a more highly resolved image. The brightness of an image alsoincreases with higher numerical aperture. High numerical aperture lensesare commercially available from a variety of vendors, and any one lenshaving a numerical aperture of equal to or greater than approximately0.6 can be used in the analyzer system. In some embodiments, the lenshas a numerical aperture of about 0.6 to about 1.3. In some embodiments,the lens has a numerical aperture of about 0.6 to about 1.0. In someembodiments, the lens has a numerical aperture of about 0.7 to about1.2. In some embodiments, the lens has a numerical aperture of about 0.7to about 1.0. In some embodiments, the lens has a numerical aperture ofabout 0.7 to about 0.9. In some embodiments, the lens has a numericalaperture of about 0.8 to about 1.3. In some embodiments, the lens has anumerical aperture of about 0.8 to about 12. In some embodiments, thelens has a numerical aperture of about 0.8 to about 1.0. In someembodiments, the lens has a numerical aperture of at least about 0.6. Insome embodiments, the lens has a numerical aperture of at least about0.7. In some embodiments, the lens has a numerical aperture of at leastabout 0.8. In some embodiments, the lens has a numerical aperture of atleast about 0.9. In some embodiments, the lens has a numerical apertureof at least about 1.0. In some embodiments, the aperture of themicroscope objective lens 140 is approximately 1.25.

The high numerical aperture (NA) microscope objective that is requiredtypically when performing single molecule detection through the walls orthe base of the sample plate 170 has short working distances. Theworking distance is the distance from the front of the lens to theobject in focus. The objective in some embodiments must be within 350microns of the object. In some embodiments, where a microscope objectivelens 140 with NA of 0.8 is used, an Olympus 40×/0.8 NA water immersionobjective (Olympus America, Inc., USA) can be used. This objective has a3.3 mm working distance. In some embodiments, an Olympus 60×/0.9 NAwater immersion objective with a 2 mm working distance can be used.Because the later lens is a water immersion lens, the space 142 betweenthe objective and the sample must be filled with water. This can beaccomplished using a water bubbler (not shown) or some other suitableplumbing for depositing water between the objective and the base of thesample plate.

In all embodiments, the electromagnetic radiation source is set so thatthe wavelength of the laser is sufficient to excite the fluorescentlabel attached to the particle. In some embodiments, the electromagneticradiation source 110 is a laser that emits light in the visiblespectrum. In some embodiments, the laser is a continuous wave laser witha wavelength of 639 nm. In other embodiments, the laser is a continuouswave laser with wavelength of 532 nm. In other embodiments, the laser isa continuous wave laser with a wavelength of 422 nm. In otherembodiments, the laser is a continuous wave laser with a wavelength of405 nm. Any continuous wave laser with a wavelength suitable forexciting a fluorescent moiety as used in the methods and compositions ofthe invention can be used without departing from the scope of theinvention.

In a single molecule analyzer system 100, as the interrogation spacepasses over the labeled single molecule, the beam 126 of theelectromagnetic radiation source directed into the interrogation spacecauses the label to enter an excited state. When the particle relaxesfrom its excited state, a detectable burst of light is emitted. In thelength of time it takes for the interrogation spice to pass over theparticle, the excitation-emission cycle is repeated many times by eachparticle. This allows the analyzer system 100 to detect tens tothousands of photons for each particle as the interrogation space passesover the particle. Photons emitted by the fluorescent particles areregistered by the detector 184 with a time delay indicative of the timefor the interrogation space to pass over the labeled particle. Thephoton intensity is recorded by the detector 184 and the sampling timeis divided into bins, wherein the bins are uniform, arbitrary timesegments with freely selectable time channel widths. The number ofsignals contained in each bin is evaluated. One or more of severalstatistical analytical methods are used to determine when a particle ispresent. These methods include determining the baseline noise of theanalyzer system and determining signal strength for the fluorescentlabel at a statistical level above baseline noise to mitigate falsepositive signals from the detector.

1. Electromagnetic Radiation Source

Some embodiments of the analyzer system use a chemiluminescent label.These embodiments may not require an EM source for particle detection.In other embodiments, the extrinsic label or intrinsic characteristic ofthe particle is light-interacting, such as a fluorescent label orlight-scattering label. In such an embodiment, a source of EM radiationis used to illuminate the label and/or the particle. EM radiationsources for excitation of fluorescent labels are preferred.

In some embodiments, the analyzer system consists of an electromagneticradiation source 110. Any number of radiation sources can be used in ascanning analyzer system 100 without departing from the scope of theinvention. Multiple sources of electromagnetic radiation have beenpreviously disclosed and are incorporated by reference from previousU.S. patent application Ser. No. 11/048,660. In some embodiments,different continuous wave electromagnetic (EM) radiation sources emitelectromagnetic radiation at the same wavelengths. In other embodiments,different sources emit different wavelengths of EM radiation.

In one embodiment, the EM source 110 is a continuous wave laserproducing wavelengths of between 200 n and 1000 nm. Continuous wavelasers provide continuous illumination without accessory electronic ormechanical devices, such as shutters, to interrupt their illumination.Such EM sources have the advantage of being small, durable andrelatively inexpensive. In addition, they generally have the capacity togenerate larger fluorescent signals than other light sources. Specificexamples of suitable continuous wave EM sources include, but are notlimited to: lasers of the argon, krypton, helium-neon, helium-cadmiumtypes, as well as, tunable diode lasers (red to infrared regions), eachwith the possibility of frequency doubling. In an embodiment where acontinuous wave laser is used, an electromagnetic radiation source of 3mW may have sufficient energy to excite a fluorescent label A beam ofsuch energy output can be between 2 to 5 μm in diameter. When exposed at3 mW, a labeled particle can be exposed to the laser beam for about 1msec. In alternate embodiments, the particle can be exposed to the laserbeam at equal to or less than about 500 μsec. In an alternateembodiment, the time of exposure can be equal to or less than about 100μsec. In an alternate embodiment, the time of exposure can be equal toor less than about 50 μsec. In an alternate embodiment, the time ofexposure can be equal to or less than about 10 μsec.

Light-emitting diodes (LEDs) are another low-cost, highly reliableillumination source. Advances in ultra-bright LEDs and dyes with highabsorption cross-section and quantum yield have made LEDs applicable forsingle molecule detection. Such LED light can be used for particledetection alone or in combination with other light sources such asmercury arc lamps, elemental arc lamps, halogen lamps, arc discharges,plasma discharges, and any combination of these.

In one embodiment, the EM source comprises a pulse wave laser. In suchan embodiment, the pulse size, size, focus spot, and total energyemitted by the laser must be sufficient to excite the fluorescent label.In some embodiments, a laser pulse of less than 1 nanosecond can beused. A pulse of this duration can be preferable in some pulsed laserapplications. In other embodiments, a laser pulse of 1 nanosecond can beused. In other embodiments, a laser pulse of 2 nanoseconds can be used.In other embodiments, a laser pulse of 3 nanoseconds can be used. Inother embodiments, a laser pulse of 4 nanoseconds can be used. In otherembodiments, a laser pulse of 5 nanoseconds can be used. In still otherembodiments, a pulse of between 2 to 5 nanoseconds can be used. In otherembodiments, a pulse of longer duration can be used.

The optimal laser intensity depends on the photo bleachingcharacteristics of the single dyes and the length of time required totraverse the interrogation space (including the speed of the particle,the distance between interrogation spaces if more than one is used andthe size of the interrogation space(s)). To obtain a maximal signal, thesample can be illuminated at the highest intensity that will not photobleach a high percentage of the dyes. The preferred intensity is suchthat no more that 5% of the dyes are bleached by the time the particlehas traversed the interrogation space.

The power of the laser is set depending on the type of dye molecules andthe length of time the dye molecules are stimulated. The power can alsodepend on the speed that the interrogation space passes through thesample. Laser power is defined as the rate at which energy is deliveredby the beam and is measured in units of Joules/second, or Watts. Toprovide a constant amount of energy to the interrogation space as theparticle passes through, the less time the laser can illuminate theparticle as the power output of the laser is increased. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is more than about 0.1, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is less than about0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100, or 110 microJoule. In some embodiments, thecombination of laser power and illumination time is such that the totalenergy received by the interrogation space during the time ofillumination is between about 0.1 and 100 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is between about 1 and 100 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe illumination time is between about 1 and 50 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is between about 2 and 50 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is between about 3 and 60 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is between about 3 and 50 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is between about 3 and 40 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is between about 3 and 30 microJoule. In someembodiments, the combination of laser power and illumination time issuch that the total energy received by the interrogation space duringthe time of illumination is about 1 microJoule. In some embodiments, thecombination of laser power and illumination time is such that the totalenergy received by the interrogation space during the time ofillumination is about 3 microJoule. In some embodiments, the combinationof laser power and illumination time is such that the total energyreceived by the interrogation space during the time of illumination isabout 5 microJoule. In some embodiments, the combination of laser powerand illumination time is such that the total energy received by theinterrogation space during the time of illumination is about 10microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 15microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 20microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 30microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 40microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 50microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation spate during the time of illumination is about 60microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 70microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 80microJoule. In some embodiments, the combination of laser power andillumination time is such that the total energy received by theinterrogation space during the time of illumination is about 90microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 100microJoule.

In some embodiments, the laser power output is set to at least about 1mW, 2 mW, 3 mW, 4 mW, 5 mW, 6 mW, 7 mW, 8 mW, 9 mW, 10 mW, 13 mW, 15 mW,20 mW, 25 mW, 30 mW, 40 mW, 50 mW, 60 mW, 70 mW, 80 mW, 90 mW, 100 mW,or more than 100 mW. In some embodiments, the laser power output is setto at least about 1 mW. In some embodiments, the laser power output isset to at least about 3 mW. In some embodiments, the laser power outputis set to at least about 5 mW. In some embodiments, the laser poweroutput is set to at least about 10 mW. In some embodiments, the laserpower output is set to at least about 15 mW. In some embodiments, thelaser power output is set to at least about 20 mW. In some embodiments,the laser power output is set to at least about 30 mW. In someembodiments, the laser power output is set to at least about 40 mW. Insome embodiments, the laser power output is set to at least about 50 mW.In some embodiments, the laser power output is set to at least about 60mW. In some embodiments, the laser power output is set to at least about90 mW.

The time that the laser illuminates the interrogation space can be setto no less than about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or1000 microseconds. The time that the laser illuminates the interrogationspace can be set to no more than about 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, 1000, 1500, or 2000 microseconds. The time that the laserilluminates the interrogation space can be set between about 1 and 1000microseconds. The time that the laser illuminates the interrogationspace can be set between about 5 and 500 microseconds. The time that thelaser illuminates the interrogation space can be set between about 5 and100 microseconds. The time that the laser illuminates the interrogationspace can be set between about 10 and 100 microseconds. The time thatthe laser illuminates the interrogation space can be set between about10 and 50 microseconds. The time that the laser illuminates theinterrogation space can be set between about 10 and 20 microseconds. Thetime that the laser illuminates the interrogation space can be setbetween about 5 and 50 microseconds. The time that the laser illuminatesthe interrogation space can be set between about 1 and 100 microseconds.In some embodiments, the time that the laser illuminates theinterrogation space is about 1 microsecond. In some embodiments, thetime that the laser illuminates the interrogation space is about 5microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 10 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 25microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 50 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 100microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 250 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 500microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 1000 microseconds.

In some embodiments, the laser illuminates the interrogation space for 1millisecond, 250 microseconds, 100 microseconds, 50 microseconds, 25microseconds or 10 microseconds with a laser that provides a poweroutput of 3 mW, 4 mW, 5 mW, or more than 5 mW. In some embodiments, alabel is illuminated with a laser that provides a power output of 3 mWand illuminates the label for about 1000 microseconds. In otherembodiments, a label is illuminated for less than 1000 milliseconds witha laser providing a power output of not more than about 20 mW. In otherembodiments, the label is illuminated with a laser power output of 20 mWfor less than or equal to about 250 microseconds. In some embodiments,the label is illuminated with a laser power output of about 5 mW forless than or equal to about 1000 microseconds.

2. Optical Scanning System

The scanning analyzer system described herein is different thantraditional single molecule analyzers previously described elsewhere. Inflow cytometry and other methods of fluorescence spectroscopy, a sampleflows through an interrogation space. In contrast, the interrogationspace in the analyzer provided herein is moved relative to the sample.This can be done by fixing the sample container relative to theinstrument and moving the electromagnetic radiation beam. Alternatively,the electromagnetic radiation beam can be fixed and the sample platemoved relative to the beam. In some embodiments, a combination of bothcan be used. In an embodiment wherein the sample plate is translated tocreate the moveable interrogation space, the limiting factor is theability to move the plate smoothly enough so that the sample located onthe sample plate is not jarred and the interrogation space is in thedesired location.

In one embodiment, the electromagnetic radiation source 110 is focusedonto a sample plate 170 of the analyzer system 100. The beam 126 fromthe continuous wave electromagnetic radiation source 110 is opticallyfocused through the base of the sample plate to a specified depth planewithin the sample located on the sample plate 170. Optical scanning ofthe sample can be accomplished using mirrors or lenses. In someembodiments, a mirror 122 is mounted on the end of a scan motor shaft124 of the scan motor 120 but is tilted at a slight angle relative tothe shaft 124. In some embodiments, as the mirror 122 turns, it candeflect the electromagnetic radiation beam 126 thereby creating a smallcircle. By placing the mirror 122 between the objective 140 and thedichroic mirror 160, the spot at the focus of the objective can movearound the sample. In some embodiments, the sample is scanned in acircular pattern. In such an embodiment, a scan circle with a diameterof between about 500 μm and about 750 μm can be formed. In someembodiments, a scan circle with a diameter of between about 550 μm and700 μm can be formed. In some embodiments, a scan circle with a diameterof between about 600 μm and 650 μm can be formed. In some embodiments ascan circle with a diameter of about 630 μm can be formed. In someembodiments, when a scan circle with a diameter of 630 μm is used, thescan circle can be traversed at about 8 revolutions per second (or about500 RPM), equivalent to pumping the sample through a flow source at arate of about 5 μl/min. In some embodiments, the scan speed of theinterrogation space is more than 100 RPM. In some embodiments, the scanspeed of the interrogation space is more than 300 RPM. In someembodiments, the scan speed of the interrogation space is more than 500RPM. In some embodiments, the scan speed of the interrogation space ismore than 700 RPM. In some embodiments, the scan speed of theinterrogation space is more than 900 RPM. In some embodiments, the scanspeed of the interrogation space is less than 1000 RPM. In someembodiments, the scan speed of the interrogation space is less than 800RPM. In some embodiments, the scan speed of the interrogation space isless than 600 RPM. In some embodiments, the scan speed of theinterrogation space is less than 400 RPM. In some embodiments, the scanspeed of the interrogation space is less than 200-RPM. In someembodiments, the scan speed of the interrogation space is between about100 RPM and about 1000 RPM. In some embodiments, the scan speed of theinterrogation space is between about 200 RPM and about 900 RPM. In someembodiments, the scan speed of the interrogation space is between about300 RPM and about 800 RPM. In some embodiments, the scan speed of theinterrogation space is between about 400 RPM and about 700 RPM. In someembodiments, the scan speed of the interrogation space is between about450 RPM and about 600 RPM. In some embodiments, the scan speed of theinterrogation space is between about 450 RPM and about 550 RPM. With thedevelopment of improved electronics and optics, scanning in the z-axismay be required in addition to scanning in a two-dimensional pattern toavoid duplicate scanning of the same molecule. In some of theembodiments previously mentioned, the optical scanning pattern allowsthe scanning of a substantially different volume each time a portion ofthe sample is scanned.

In some embodiments, the sample is scanned by an electromagneticradiation source wherein the electromagnetic radiation interrogates aportion of the sample. A single molecule of interest may or may not bepresent in the interrogation space. In some embodiments, a portion ofthe sample is scanned a first time and then subsequently scanned asecond time. In some embodiments the same portion of sample is scannedmultiple times. In some embodiments, the sample is scanned such that thedetection spot returns to a portion of sample a second time aftersufficient time has passed so that the molecules detected in the firstpass have drifted or diffused out of the portion, and other moleculeshave drifted or diffused into the portion. When the same portion ofsample is scanned at least one or more times, the scanning speed can beslow enough to allow molecules to diffuse into, and out of, the spacebeing interrogated. In some embodiments, the interrogation space istranslated through a same portion of sample a first time and a secondtime at a sufficiently slow speed as to allow a molecule of interestthat is detected the first time the interrogation space is translatedthrough the portion of sample to substantially diffuse out of theportion of sample after the first time the portion of sample isinterrogated by the interrogation space, and to further allow asubsequent molecule of interest, if present, to substantially diffuseinto the portion of sample the second time the portion of sample isinterrogated by the interrogation space. FIG. 2 shows a graph of thediffusion time versus corresponding diffusion radius for molecules witha 155 KDa molecular weight. As used herein, “diffusion radius” refers tothe standard deviation of the distance from the starting point that themolecule will most likely diffuse in the time indicated on the X-axis.

In some embodiments an alternative scan pattern is used. In someembodiments, the scan pattern can approximate an arc. In someembodiments, the scan pattern comprises at least one 90 degree angle. Insome embodiments, the scan pattern comprises at least one angle lessthan 90 degrees. In some embodiments, the scan pattern comprises atleast one angle that is more than 90 degrees. In some embodiments, thescan pattern is substantially sinusoidal. In some embodiments, theoptical scanning can be done with one mirror as previously described. Inan alternative embodiment, the optical scanning can be done with atleast two mirrors. Multiple mirrors allow scanning in a straight line,as well as allowing the system to scan back and forth, so that aserpentine pattern is created. Alternatively, a multiple mirror opticalscanning configuration allows for scanning in a raster pattern.

In an alternative embodiment, optical scanning can be done using anoptical wedge. A wedge scanner provides a circular scan pattern andshortens the optical path because scan lenses are not required. Anoptical wedge approximates a prism with a very small angle. The opticalwedge can be mounted to the shaft of the electromagnetic radiationsource. The optical wedge rotates to create an optical scan pattern. Inan alternative embodiment, the scan mirror can be mounted using anelectro-mechanical mount. In such an embodiment, the electro-mechanicalmount would have two voice coils. One voice coil would causedisplacement of the mirror in a vertical direction. The other voice coilwould cause displacement of the mirror in a horizontal direction. Usingthis embodiment, any scan pattern desired can be created.

In some embodiments, the scanning particle analyzer scans the samplelocated in the sample plate in a two-dimensional orientation, e.g.,following the x-y plane of the sample. In some embodiments, the samplecan be scanned in a three-dimensional orientation consisting of scanningin an x-y plane and z direction. In some embodiments, the sample can bescanned along the x-y and z directions simultaneously. For example, thesample can be scanned in a helical pattern. In some embodiments, thesample can be scanned in the z direction only.

In some embodiments, a scan lens (130 as shown in FIGS. 1A & 1B) canre-direct the scanning optical path to the pupil of the objective. Thescan lens focuses the image of the optical axis on the scan mirror tothe exit pupil of the objective. The scan lens ensures that the scanningbeam remains centered on the objective, despite the distance between thescan mirror and the microscope objective, thus improving the image andlight collection efficiency of the scanning beam.

3. Interrogation Space

The invention described herein encompasses the use of an interrogationspace, which can be thought of as an effective volume of sample in whicha single molecule of interest can be detected when present. Althoughthere are various ways to calculate the interrogation space of thesample, the simplest method for determining the effective volume (V) ofthe interrogation space is to calculate the effective cross section ofthe detection volume. Because the detection volume is typically sweptthrough the sample by translating the detection volume through thestationary sample, the volume is typically the result of the crosssectional area of the detection volume being swept through some distanceduring the time of measurement. If the sample concentration (C) is knownand the number of molecules detected (N) during a period of time isknown, then the sample volume consists of the number of moleculesdetected divided by the concentration of the sample, or V=N/C (where thesample concentration has units of molecules per unit volume).

For example, in some embodiments of the system described herein, allphotons detected are counted and added up in 1 msec segments (photoncounting bins). If a molecule of interest is present in the 1 msecsegment, the count of photons detected is typically significantly higherthan background. Therefore, the distance the detection volume has movedwith respect to the sample is the appropriate distance to use tocalculate the volume sampled in a single segment, i.e., theinterrogation space. In this example, if the sample is analyzed for 60seconds, then effectively 60,000 segments are scanned. If the effectivevolume is divided by the number of segments, the resulting volume is inessence the volume of a single segment, i.e., the interrogation space.Mathematically, the volume of the single segment, i.e., theinterrogation space volume (Vs), equals the number of molecules detected(N) divided by the concentration of the sample multiplied by the numberof segment bins (C·n—where n represents the number of segment binsduring the time the N number of molecules were counted). For exemplarypurposes only, consider that a known standard of one femtomolarconcentration is run through 60,000 segments, and 20 molecules of thestandard are detected. Accordingly, the interrogation space volume, Vs,equals N/(C·n) or 20/(602.214·6E4), or 553.513 μm³. Thus, in thisexample, the interrogation space volume, which is the effective volumefor one sample corresponding to one photon counting bin, is 553.513 μm³.

In addition, from the interrogation volume described previously, thecross sectional area of the sample segment can be approximated using acapillary flow system with similar optics to the invention describedherein. The cross section area (A) is approximated by dividing theinterrogation volume (Vs) by the distance (t) the detection segmentmoves. The distance (t) the detection segment moves is given by i·r·s/x,where t a function of the flow rate (r), the viscosity of the sample(i), the segment bin time (s), and the cross section of the capillary(x). For exemplary purposes only, consider a bin time (s) of 1 msec, aflow rate (r) of 5 μL/min, a viscosity factor (i) of 2, and a capillarycross sectional area (x) of 10,000 μm². Accordingly, the distance theinterrogation space moves (t) is given by i·r·s/x, or (2·5 μL/min·1E-3sec)/(10,000 μm²), or 16.7 μm. The effective cross sectional area (A) ofthe detector spot can further be calculated as Vs/t, or (553.513μm³)/(16.7 μm), or 33 μm². Note that both the value of the interrogationvolume, Vs, and the cross sectional area of the interrogation volumedepend on the binning time.

The lower limit on the size of the interrogation space is bounded by thewavelengths of excitation energy currently available. The upper limit ofthe interrogation space size is determined by the desiredsignal-to-noise ratios—the larger the interrogation space, the greaterthe noise from, e.g., Raman scattering. In some embodiments, the volumeof the interrogation space is more than about 1 μm³, more than about 2μm³, more than about 3 μm³, more than about 4 μm³, more than about 5μm³, more than about 10 μm³, more than about 15 μm³, more than about 30μm³, more than about 50 μm³, more than about 75 μm³, more than about 100μm³, more than about 150 μm³, more than about 200 μm³, more than about250 μm³, more than about 300 μm³, more than about 400 μm³, more thanabout 500 μm³, more than about 550 μm³, more than about 600 μm³, morethan about 750 μm³, more than about 1000 μm³, more than about 2000 μm³,more than about 4000 μm³, more than about 6000 μm³, more than about 8000μm³, more than about 10000 μm³, more than about 12000 μm³, more thanabout 13000 μm³, more than about 14000 μm³, more than about 15000 μm³,more than about 20000 μm³, more than about 30000 μm³, more than about40000 μm³, or more than about 50000 μm³. In some embodiments, theinterrogation space is of a volume less than about 50000 μm³, less thanabout 40000 μm³, less than about 30000 μm³, less than about 20000 μm³,less than about 15000 μm³, less than about 14000 μm³, less than about13000 μm³, less than about 12000 μm³, less than about 11000 μm³, lessthan about 9500 μm³, less than about 8000 μm³, less than about 6500 μm³,less than about 6000 μm³, less than about 5000 μm³, less than about 4000μm³, less than about 3000 μm³, less than about 2500 μm³, less than about2000 μm³, less than about 1500 μm³, less than about 1000 μm³, less thanabout 800 μm³, less than about 600 μm³, less than about 400 μm³, lessthan about 200 μm³, less than about 100 μm³, less than about 75 μm³,less than about 50 μm³, less than about 25 μm³, less than about 20 μm³,less than about 15 μm³, less than about 14 μm³, less than about 13 μm³,less than about 12 μm³, less than about 11 μm³, less than about 10 μm³,less than about 5 μm³, less than about 4 μm³, less than about 3 μm³,less than about 2 μm³, or less than about 1 μm³. In some embodiments,the volume of the interrogation space is between about 1 μm³ and about10000 μm³. In some embodiments, the interrogation space is between about1 μm³ and about 1000 μm³. In some embodiments, the interrogation spaceis between about 1 μm³ and about 100 μm³. In some embodiments, theinterrogation space is between about 1 μm³ and about 50 μm³. In someembodiments, the interrogation space is between about 1 μm³ and about 10μm³. In some embodiments, the interrogation space is between about 2 μm³and about 10 μm³. In some embodiments, the interrogation space isbetween about 3 μm³ and about 7 μm³.

4. Sample Plate

Some embodiments of the invention described herein use a sample plate170 to hold the sample being detected for a single molecule of interest.The sample plate in some embodiments is a microtiter plate. Themicrotiter plate consists of a base 172 and a top surface 174. The topsurface 174 of the microtiter plate in some embodiments consists of atleast one well for containing a sample of interest. In some embodiments,the microtiter plate consists of a plurality of wells to contain aplurality of samples. The system described herein is sensitive enough sothat only a small sample size is needed. In some embodiments the samplesize can be less than approximately 100 μl. In some embodiments, thesample size can be less than approximately 10 μl. In some embodiments,the sample size can be less than approximately 1 μl. In someembodiments, the sample is less than approximately 0.1 μl. In someembodiments, the sample size is less than approximately 0.001 μl. Themicrotiter plate in some embodiments can be one constructed usingmicrofabrication techniques. In some embodiments, the top surface of theplate can be smooth. The sample can be sized so that the sample isself-contained by the surface tension of the sample itself. In such anembodiment, the sample forms a droplet on the surface of the plate. Insome embodiments, the sample can then be scanned for a molecule ofinterest.

Typically, the sample is scanned through the sample plate material,e.g., through the walls of the microwells. In some embodiments, thesample is scanned through the base of the sample plate. In someembodiments, the base of the sample plate is made of a material that istransparent to light. In some embodiments, the base of the sample plateis made of a material that is transparent to electromagnetic radiation.The sample plate is transparent to an excitation wavelength of interest.Using a transparent material allows the wavelength of the excitationbeam to pass through the sample plate and excite the molecule ofinterest or the fluorescent label conjugated to the molecule ofinterest. The transparency of the plate further allows the detector todetect the emissions from the excited molecules of interest. In someembodiments, the base material is substantially transparent to light ofwavelengths between 550 nm and 800 nm. In some embodiments, the basematerial is substantially transparent to light of wavelengths between600 nm and 700 nm. In some embodiments, the material of the plate istransparent to light of wavelength between 620 nm and 680 nm. In someembodiments, the material of the plate is transparent to light ofwavelengths between 630 nm and 660 nm. In some embodiment, the materialof the plate is transparent to light of wavelength between 630 nm and640 nm.

The thickness of the sample plate is also considered. The sample isscanned by an electromagnetic radiation source that passes through aportion of the material of the plate. The thickness of the plate allowsan image to be formed on a first side of the portion of the plate thatis scanned by a high numerical aperture lens that is positioned on asecond side of the portion of the plate that is scanned. Such anembodiment facilitates the formation of an image within the sample andnot within the base. The image formed corresponds to the interrogationspace of the system. The image should be formed at the depth of thesingle molecule of interest. As previously mentioned, the thickness ofthe plate depends on the working distance and depth of field of the lensthat is used. Commercial plates available are typically 650 micronsthick.

The plate can be made out of any suitable material that allows theexcitation energy to pass through the plate. In some embodiments theplate is made of polystyrene. In some embodiments, the plate is made ofpolycarbonate. In some embodiments, the plate is made of polyethylene.In some embodiments, a commercially available plate can be used, such asa NUNC™ brand plate. Any plate made of a suitable material and of asuitable thickness can be used. In preferred embodiments, the plate ismade out of a material with low fluorescence, thereby reducingbackground fluorescence. For example, a preferred material may emit lessfluorescence than a plate made from polystyrene. Background fluorescenceresulting from the plate material can be further avoided by minimizingthe thickness of the plate.

In some embodiments, the sample consists of a small volume of fluid thatcan contain a particular type of molecule. In such an embodiment, thesingle molecule of interest, if present, can be detected and counted ina location anywhere in the fluid volume. In some embodiments, scanningthe sample comprises scanning a smaller concentrated sample. In such anembodiment, the optical scanning can occur at the surface of the sampleplate, for example, if the highest concentration of molecules is locatedat the surface of the sample plate. This can occur if the singlemolecules are adsorbed to the surface of the plate or if they are boundto antibodies or other binding molecules adhered to the surface of theplate. When antibodies are used to capture a single molecule ofinterest, the antibodies can be applied to the surface of the sampleplate, e.g., to the bottom of a microwell(s). The single molecule ofinterest then binds to the antibodies located within the microwell. Insome embodiments, an elution step is done to remove the bound singlemolecule of interest. The presence or absence of the unbound moleculescan then be detected in a smaller sample volume. In some embodimentswherein the elution step is done, the single molecules may or may not beattached to paramagnetic beads. If no beads are used, the elution buffercan be added to the sample well and the presence or absence of thesingle molecule of interest can be detected. In some embodiments, aparamagnetic bead is used as a capture bead to capture the singlemolecule of interest.

In some embodiments of the scanning single molecule analyzer describedherein, the electromagnetic (EM) radiation source is directed to thesample interrogation space without passing through the material of thesample plate. Image formation occurs in the sample on the same side asthe beam directed to the sample. In such an embodiment, a waterimmersion lens can be used but is not required to image the samplethrough the air-liquid interface. In zero carryover systems wherein theobjective does not come in contact with the sample, sample carryoverbetween samples does not occur.

5. Detectors

In one embodiment, light emitted by a fluorescent label after exposureto electromagnetic radiation is detected. The emitted light can be,e.g., ultra-violet, visible or infrared. Referring to FIGS. 1A & 1B, thedetector 184 (or other embodiments) can capture the amplitude andduration of photon bursts from a fluorescent moiety, and convert theamplitude and duration of the photon bursts to electrical signals.Detection devices such as CCD cameras, video input module cameras, andStreak cameras can be used to produce images with contiguous signals.Other embodiments use devices such as a bolometer, a photodiode, aphotodiode array, avalanche photodiodes, and photomultipliers whichproduce sequential signals. Any combination of the aforementioneddetectors can be used.

Several distinct characteristics of the emitted electromagneticradiation between an interrogation space and its corresponding detector180, can be detected including: emission wavelength, emission intensity,burst size, burst duration, and fluorescence polarization. In someembodiments, the detector 180 is a photodiode used in reverse bias. Sucha photodiode set usually has an extremely high resistance. Thisresistance is reduced when light of an appropriate frequency shines onthe P/N junction. Hence, a reverse biased diode can be used as adetector by monitoring the current running through it. Circuits based onthis effect are more sensitive to light than circuits based on zerobias.

In one embodiment of the analyzer system, the photodiode can be anavalanche photodiode. These photodiodes can be operated with much higherreverse bias than conventional photodiodes, thus allowing eachphoto-generated carrier to be multiplied by avalanche breakdown. Thisresults in internal gain within the photodiode, thereby increasing theeffective responsiveness and sensitivity of the device. The choice ofphotodiode is determined by the energy or emission wavelength emitted bythe fluorescently labeled particle. In some embodiments, the detector isan avalanche photodiode detector that detects energy between 300 nm and1700 nm. In another embodiment, silicon avalanche photodiodes can beused to detect wavelengths between 300 nm and 1100 nm. In anotherembodiment, the photodiode is an indium gallium arsenide photodiode thatdetects energy in the range of 800-2600 nm. In another embodiment,indium gallium arsenic photodiodes can be used to detect wavelengthsbetween 900 nm and 1700 nm. In some embodiments, the photodiode is asilicon photodiode that detects energy in the range of 190-1100 nm. Inanother embodiment, the photodiode is a germanium photodiode thatdetects energy in the range of 800-1700 nm. In yet other embodiments,the photodiode is a lead sulfide photodiode that detects energy in therange of between less than 1000 nm to 3500 nm. In some embodiments, theavalanche photodiode is a single-photon detector designed to detectenergy in the 400 nm to 1100 nm wavelength range. Single photondetectors are commercially available (for example Perkin Elmer,Wellesley, Mass.).

In some embodiments, an analyzer system can comprise at least onedetector. In other embodiments, the analyzer system can comprise atleast two detectors, and each detector can be chosen and configured todetect light energy at a specific wavelength range. For example, twoseparate detectors can be used to detect particles tagged with differentlabels, which emit photons with energy in different spectra uponexcitation with an EM source. In one embodiment, an analyzer system cancomprise a first detector that can detect fluorescent energy in therange of 450-700 nm such as that emitted by a green dye (e.g., AlexaFluor 546), and a second detector that can detect fluorescent energy inthe range of 620-780 nm such as that emitted by a far-red dye (e.g.,Alexa Fluor 647). Other embodiments use detectors for detectingfluorescent energy in the range of 400-600 nm such as that emitted byblue dyes (e.g., Hoechst 33342), and for detecting energy in the rangeof 560-700 nm such as that emitted by red dyes (e.g., Alexa Fluor 546and Cy3).

A system comprising two or more detectors can be used to detectindividual particles that are each tagged with two or more labelsemitting light in different spectra. For example, two differentdetectors can detect an antibody that has been tagged with two differentdye labels. Alternatively, an analyzer system comprising two detectorscan be used to detect particles of different types, each type beingtagged with a different dye molecule, or with a mixture of two or moredye molecules. For example, two different detectors can be used todetect two different types of antibodies that recognize two differentproteins, each type being tagged with a different dye label or with amixture of two or more dye label molecules. By varying the proportion ofthe two or more dye label molecules, two or more different particletypes can be individually detected using two detectors. It is understoodthat three or more detectors can be used without departing from thescope of the invention.

It should be understood by one skilled in the art that one or moredetectors can be configured at each interrogation space, whether one ormore interrogation spaces are defined within a flow cell, and that eachdetector can be configured to detect any of the characteristics of theemitted electromagnetic radiation listed above. The use of multipledetectors, e.g., for multiple interrogation spaces, has been previouslydisclosed in a prior application and is incorporated by reference hereinfrom U.S. patent application Ser. No. 11/048,660. Once a particle islabeled to render it detectable (or if the particle possesses anintrinsic characteristic rendering it detectable), any suitabledetection mechanism known in the art can be used without departing fromthe scope of the present invention, for example a CCD camera, a videoinput module camera, a Streak camera, a bolometer, a photodiode, aphotodiode array, avalanche photodiodes, and photomultipliers producingsequential signals, and combinations thereof. Different characteristicsof the electromagnetic radiation can be detected including: emissionwavelength, emission intensity, burst size, burst duration, fluorescencepolarization, and any combination thereof.

III. Molecules for Single Molecule Detection

The instruments, kits and methods of the invention can be used for thesensitive detection and determination of concentration of a number ofdifferent types of single molecules. In particular, the instruments,kits, and methods are useful in the sensitive detection anddetermination of concentration of markers of biological states.“Detection of a single molecule,” as that term is used herein, refers toboth direct and indirect detection. For example, a single molecule canbe labeled with a fluorescent label, and the molecule-label complexdetected in the instruments described herein. Alternatively, a singlemolecule can be labeled with a fluorescent label, then the fluorescentlabel is detached from the single molecule, and the label detected inthe instruments described herein. The term detection of a singlemolecule encompasses both forms of detection.

A. General

Examples of molecules that can be detected using the analyzer andrelated methods of the present invention include: biopolymers such asproteins, nucleic acids, carbohydrates, and small molecules, bothorganic and inorganic. In particular, the instruments, kits, and methodsdescribed herein are useful in the detection of single molecules ofproteins and small molecules in biological samples, and thedetermination of concentration of such molecules in the sample.

The terms “protein,” “polypeptide,” “peptide,” and “oligopeptide,” areused interchangeably herein and include any composition that includestwo or more amino acids joined together by a peptide bond. It will beappreciated that polypeptides can contain amino acids other than the 20amino acids commonly referred to as the 20 naturally occurring aminoacids. Also, polypeptides can include one or more amino acids, includingthe terminal amino acids, which are modified by any means known in theart (whether naturally or non-naturally). Examples of polypeptidemodifications include e.g., by glycosylation, orother-post-translational modification. Modifications which can bepresent in polypeptides of the present invention, include, but are notlimited to: acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a polynucleotide or polynucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, OP anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

The molecules detected by the present instruments, kits, and methods canbe free or can be part of a complex, e.g., an antibody-antigen complex,or more generally a protein-protein complex, e.g., complexes of troponinor complexes of prostate specific antigen (PSA).

B. Markers of Biological States

In some embodiments, the invention provides compositions and methods forthe sensitive detection of biological markers, and for the use of suchmarkers in diagnosis, prognosis, and/or determination of methods oftreatment.

Markers of the present invention can be, for example, any compositionand/or molecule or a complex of compositions and/or molecules that isassociated with a biological state of an organism (e.g., a conditionsuch as a disease or a non-disease state). A marker can be, for example,a small molecule, a polypeptide, a nucleic acid, such as DNA and RNA, alipid, such as a phospholipid or a micelle, a cellular component such asa mitochondrion or chloroplast, etc. Markers contemplated by the presentinvention can be previously known or unknown. For example, in someembodiments, the methods herein can identify novel polypeptides that canbe used as markers for a biological state of interest or condition ofinterest, while in other embodiments, known polypeptides are identifiedas markers for a biological state of interest or condition. Using thesystems of the invention it is possible that one can observe thosemarkers, e.g., polypeptides with high potential use in determining thebiological state of an organism, but that are only present at lowconcentrations, such as those “leaked” from diseased tissue. Other highpotentially useful markers or polypeptides can be those that are relatedto the disease, for instance, those that are generated in the tumor-hostenvironment. Any suitable marker that provides information regarding abiological state can be used in the methods and compositions of theinvention. A “marker,” as that term is used herein, encompasses anymolecule that can be detected in a sample from an organism and whosedetection or quantitation provides information about the biologicalstate of the organism.

Biological states include but are not limited to phenotypic states;conditions affecting an organism; states of development; age; health;pathology; disease detection, process, or staging; infection; toxicity;or response to chemical, environmental, or drug factors (such as drugresponse phenotyping, drug toxicity phenotyping, or drug effectivenessphenotyping).

The term “organism” as used herein refers to any living being comprisedof a least one cell. An organism can be as simple as a one cell organismor as complex as a mammal. An organism of the present invention ispreferably a mammal. Such mammal can be, for example, a human or ananimal such as a primate (e.g., a monkey, chimpanzee, etc.), adomesticated animal (e.g., a dog, cat, horse, etc.), farm animal (e.g.,goat, sheep, pig, cattle, etc.), or laboratory animal (e.g., mouse, rat,etc.). Preferably, an organism is a human.

In some embodiments, the methods and compositions of the invention aredirected to classes of markers, e.g., cytokines, growth factors,oncology markers, markers of inflammation, endocrine markers, autoimmunemarkers, thyroid markers, cardiovascular markers, markers of diabetes,markers of infectious disease, neurological markers, respiratorymarkers, gastrointestinal markers, musculoskeletal markers,dermatological disorders, and metabolic markers.

Table 1, below, provides examples of these classes of markers that havebeen measured by the methods and compositions of the invention, andprovides the concentration of the markers as detected by the methods andcompositions of the invention and number of particles that are countedby the single molecule analyzer system of the invention for theparticular marker.

TABLE 1 CLASSES OF MARKERS AND EXEMPLARY MARKERS IN THE CLASSES MolarConc. Molecules Cytokines IL-12 p70 2.02 × 10⁻¹⁴ 6.09 × 10⁺⁵ IL-10 5.36× 10⁻¹⁴ 1.61 × 10⁺⁶ IL-1 alpha 5.56 × 10⁻¹⁴ 1.67 × 10⁺⁶ IL-3 5.85 ×10⁻¹⁴ 1.76 × 10⁺⁶ IL-12 p40 6.07 × 10⁻¹⁴ 1.83 × 10⁺⁶ IL-1ra 6.12 × 10⁻¹⁴1.84 × 10⁺⁶ IL-12 8.08 × 10⁻¹⁴ 2.44 × 10⁺⁶ IL-6 9.53 × 10⁻¹⁴ 2.87 × 10⁺⁶IL-4 1.15 × 10⁻¹³ 3.47 × 10⁺⁶ IL-18 1.80 × 10⁻¹³ 5.43 × 10⁺⁶ IP-10 1.88× 10⁻¹³ 1.13 × 10⁺⁷ IL-5 1.99 × 10⁻¹³ 5.98 × 10⁺⁶ Eotaxin 2.06 × 10⁻¹³1.24 × 10⁺⁷ IL-16 3.77 × 10⁻¹³ 1.14 × 10⁺⁷ MIG 3.83 × 10⁻¹³ 1.15 × 10⁺⁷IL-8 4.56 × 10⁻¹³ 1.37 × 10⁺⁷ IL-17 5.18 × 10⁻¹³ 1.56 × 10⁺⁷ IL-7 5.97 ×10⁻¹³ 1.80 × 10⁺⁷ IL-15 6.13 × 10⁻¹³ 1.84 × 10⁺⁷ IL-13 8.46 × 10⁻¹³ 2.55× 10⁺⁷ IL-2R (soluble) 8.89 × 10⁻¹³ 2.68 × 10⁺⁷ IL-2 8.94 × 10⁻¹³ 2.69 ×10⁺⁷ LIF/HILDA 9.09 × 10⁻¹³ 5.47 × 10⁺⁷ IL-1 beta 1.17 × 10⁻¹² 3.51 ×10⁺⁷ Fas/CD95/Apo-1 1.53 × 10⁻¹² 9.24 × 10⁺⁷ MCP-1 2.30 × 10⁻¹² 6.92 ×10⁺⁷ Oncology EGF 4.75 × 10⁻¹⁴ 2.86 × 10⁺⁶ TNF-alpha 6.64 × 10⁻¹⁴ 8.00 ×10⁺⁶ PSA (3rd generation) 1.15 × 10⁻¹³ 6.92 × 10⁺⁶ VEGF 2.31 × 10⁻¹³6.97 × 10⁺⁶ TGF-beta1 2.42 × 10⁻¹³ 3.65 × 10⁺⁷ FGFb 2.81 × 10⁻¹³ 1.69 ×10⁺⁷ TRAIL 5.93 × 10⁻¹³ 3.57 × 10⁺⁷ TNF-RI (p55) 2.17 × 10⁻¹² 2.62 ×10⁺⁸ Inflammation ICAM-1 (soluble) 8.67 × 10⁻¹⁵ 5.22 × 10⁺⁴ RANTES 6.16× 10⁻¹⁴ 3.71 × 10⁺⁶ MIP-2 9.92 × 10⁻¹⁴ 2.99 × 10⁺⁶ MIP-1 beta 1.98 ×10⁻¹³ 5.97 × 10⁺⁶ MIP-1 alpha 2.01 × 10⁻¹³ 6.05 × 10⁺⁶ MMP-3 1.75 ×10⁻¹² 5.28 × 10⁺⁷ Endocrinology 17 beta-Estradiol (E2) 4.69 × 10⁻¹⁴ 2.82× 10⁺⁶ DHEA 4.44 × 10⁻¹³ 2.67 × 10⁺⁷ ACTH 1.32 × 10⁻¹² 7.96 × 10⁺⁷Gastrin 2.19 × 10⁻¹² 1.32 × 10⁺⁸ Growth Hormone (hGH) 2.74 × 10⁻¹² 1.65× 10⁺⁸ Autoimmune GM-CSF 1.35 × 10⁻¹³ 8.15 × 10⁺⁶ C-Reactive Protein(CRP) 3.98 × 10⁻¹³ 2.40 × 10⁺⁷ G-CSF 1.76 × 10⁻¹² 1.06 × 10⁺⁸ ThyroidCyclic AMP 9.02 × 10⁻¹⁵ 5.43 × 10⁺⁵ Calcitonin 3.25 × 10⁻¹⁴ 1.95 × 10⁺⁶Parathyroid Hormone (PTH) 1.56 × 10⁻¹³ 9.37 × 10⁺⁶ CardiovascularB-Natriuretic Pepide 2.86 × 10⁻¹³ 1.72 × 10⁺⁷ NT-proBNP 2.86 × 10⁻¹²8.60 × 10⁺⁷ C-Reactive Protein, HS 3.98 × 10⁻¹³ 2.40 × 10⁺⁷Beta-Thromboglobulin (BTG) 5.59 × 10⁻¹³ 3.36 × 10⁺⁷ Diabetes C-Peptide2.41 × 10⁻¹⁵ 1.45 × 10⁺⁵ Leptin 1.89 × 10⁻¹³ 1.14 × 10⁺⁷ Infectious Dis.IFN-gamma 2.08 × 10⁻¹³ 1.25 × 10⁺⁷ IFN-alpha 4.55 × 10⁻¹³ 2.74 × 10⁺⁷Metabolism Bio-Intact PTH (1-84) 1.59 × 10⁻¹² 1.44 × 10⁺⁸ PTH 1.05 ×10⁻¹³ 9.51 × 10⁺⁶

Cytokines

For both research and diagnostics, cytokines are useful as markers of anumber of conditions, diseases, pathologies, and the like, and thecompositions and methods of the invention include labels for detectionand quantitation of cytokines and methods using such labels to determinenormal and abnormal levels of cytokines, as well as methods ofdiagnosis, prognosis, and/or determination of treatment based on suchlevels.

There are currently over 100 cytokines/chemokines whose coordinate ordiscordant regulation is of clinical interest. In order to correlate aspecific disease process with changes in cytokine levels, the idealapproach requires analyzing a sample for a given cytokine, or multiplecytokines, with high sensitivity. Exemplary cytokines that are presentlyused in marker panels and that can be used in methods and compositionsof the invention include, but are not limited to, BDNF, CREB pS133, CREBTotal, DR-5, EGF, ENA-78, Eotaxin, Fatty Acid Binding Protein,FGF-basic, granulocyte colony-stimulating factor (G-CSF), GCP-2,Granulocyte-macrophage Colony-stimulating Factor GM-CSF (GM-CSF),growth-related oncogene-keratinocytes (GRO-KC), HGF, ICAM-1, IFN-alpha,IFN-gamma, the interleukins IL-10, IL-11, IL-12, IL-12 p40, IL-12p40/p70, IL-12 p70, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1alpha,IL-1beta, IL-1ra, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, interferon-inducible protein (10 IP-10), JE/MCP-1,keratinocytes (KC), KC/GROa, LIF, Lymphotacin, M-CSF, monocytechemoattractant protein-1 (MCP-1), MCP-1 (MCAF), MCP-3, MCP-5, MDC, MIG,macrophage inflammatory (MIP-1 alpha), MIP-1 beta, MIP-1 gamma, MIP-2,MIP-3 beta, OSM, PDGF-BB, regulated upon activation, normal T cellexpressed and secreted (RANTES), Rb (pT821), Rb (total), Rb pSpT249/252,Tau (pS214), Tau (pS396), Tau (total), Tissue Factor, tumor necrosisfactor-alpha (TNF-alpha), TNF-beta, TNP-RI, TNP-RII, VCAM-1, and VEGF.In some embodiments, the cytokine is IL-12p70, IL-10, IL-1 alpha, IL-3,IL-12 p40, IL-1ra, IL-12, IL-6, IL-4, IL-18, IL-10, IL-5, eotaxin,IL-16, MIG, IL-8, IL-17, IL-7, IL-15, IL-13, IL-2R (soluble), IL-2,LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1, and MCP-1.

Growth Factors

Growth factors that can be used in methods and compositions of theinvention include EGF Ligands such as Amphiregulin, LRIG3, Betacellulin,Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen, TGF-alpha,Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1; EGF R/ErbBReceptor Family such as EGF R, ErbB3, ErbB2, ErbB4; FGF Family such asFGF LigandsFGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4,FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-22,FGF-10, FGF-23, FGF-11, KGF/FGF-7, FGF Receptors FGF R1-4, FGF R3, FGFR1, FGF R4, FGF R2, FGF R5, FGF Regulators FGF-BP; the Hedgehog FamilyDesert Hedgehog, Sonic Hedgehog, Indian Hedgehog; Hedgehog RelatedMolecules & Regulators BOC, GLI-3, CDO, GSK-3 alpha/beta, DISP1, GSK-3alpha, Gas1, GSK-3 beta, GLI-1, Hip, GLI-2; the IGF FamilyIGFLigandsIGP-1, IGF-II, IGF-I Receptor (CD221) IGF-I R, and IGF BindingProtein (IGFBP) Family ALS, IGFBP-5, CTGF/CCN2, IGFBP-6, Cyr61/CCN1,IGFBP-L1, Endocan, IGFBP-rp1/IGFBP-7, IGFBP-1, IGFBP-rP10, IGFBP-2,NOV/CCN3, IGFBP-3, WISP-1/CCN4, IGFBP-4; Receptor Tyrosine Kinases Ax1,FGF R4, C1q R1/CD93, POP R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R,EGF, R IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2, M-CSF R,EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha, EphA7,PDGF R beta, EphA8, Ret, EphB1, RTK-like Orphan Receptor 1/ROR1, EphB2,RTK-like Orphan Receptor 2/ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1,EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF, R1-4 VEGF R,FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/KDR/Flk-1, FGF R3, VEGF R3/Flt-4;Proteoglycans & Regulators Proteoglycans Aggrecan, Mimecan, Agrin,NG2/MCSP, Biglycan, Osteoadherin, Decorin, Podocan, DSPG3,delta-Sarcoglycan, Endocan, Syndecan-1/CD138, Endoglycan, Syndecan-2,Endorepellin/Perlecan, Syndecan-3, Glypican 2, Syndecan-4, Glypican 3,Testican 1/SPOCK1, Glypican 5, Testican 2/SPOCK2, Glypican 6, Testican3/SPOCK3, Lumican, Versican, Proteoglycan Regulators, ArylsulfataseA/ARSA, Glucosamine (N-acetyl)-6-Sulfatase/GNS, Exostosin-like 2/EXTL2,HS6ST2, Exostosin-like 3/EXTL3, Iduronate 2-Sulfatase/IDS, GalNAc4S-6ST;SCF, Flt-3 Ligand & M-CSF Flt-3, M-CSF R, Flt-3 Ligand, SCF, M-CSF, SCFR/c-kit; TGF-beta Superfamily (same as listed for inflammatory markers);VEGF/PDGF Family Neuropilin-1, PlGF, Neuropilin-2, PlGF-2, PDGF, VEGF,PDGF R alpha, VEGF-B, PDGF R beta, VEGF-C, PDGF-A, VEGF-D, PDGF-AB, VEGFR, PDGF-B, VEGF R1/Flt-1, PDGF-C, VEGF R2/KDR/Flk-1, PDGF-D, VEGFR3/Flt-4; Wnt-related Molecules Dickkopf Proteins & Wnt InhibitorsDkk-1,Dkk-4, Dkk-2, Soggy-1, Dkk-3, WIF-1 Frizzled & Related ProteinsFrizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1,Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7,MFRP Wnt Ligands Wnt-1, Wnt-8a, Wnt-2b, Wnt-8b, Wnt-3a, Wnt-9a, Wnt-4,Wnt-9b, Wnt-5a, Wnt-10a, Wnt-5b, Wnt-10b, Wnt-7a, Wnt-11, Wnt-7b; OtherWnt-related Molecules APC, Kremen-2, Axin-1, LRP-1, beta-Catenin, LRP-6,Dishevelled-1, Norrin, Dishevelled-3, PKC beta 1, Glypican 3, Pygopus-1,Glypican 5, Pygopus-2, GSK-3 alpha/beta, R-Spondin 1, GSK-3 alpha,R-Spondin 2, GSK-3 beta, R-Spondin 3, ICAT, RTK-like Orphan Receptor1/ROR1, Kremen-1, RTK-like Orphan Receptor 2/ROR, and Other GrowthFactors CTGF/CCN2, beta-NOF, Cyr61/CCN1, Norrin, DANCE, NOV/CCN3,EG-VEGF/PK1, Osteocrin, Hepassocin, PD-ECGF, HGF, Progranulin, LECT2,Thrombopoietin, LEDGF, and WISP-1/CCN4.

Markers of Inflammation

Markers of inflammation that can be used in methods and compositions ofthe invention include ICAM-1, RANTES, MIP-2, MIP-1-beta, MIP-1-alpha,and MMP-3. Further markers of inflammation include adhesion moleculessuch as the integrins α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α8β1,α9β1, αVβ1, α4β7, α6β4, αDβ2, αLβ2, αMβ2, αVβ3, αVβ5, αVβ6, αVβ8, αXβ2,αIIbβ3, αIELbβ7, beta-2 integrin, beta-3 integrin, beta-2 integrin,beta-4 integrin, beta-5 integrin, beta-6 integrin, beta-7 integrin,beta-8 integrin, alpha-1 integrin, alpha-2 integrin, alpha-3 integrin,alpha-4 integrin, alpha-5 integrin, alpha-6 integrin, alpha-7 integrin,alpha-8 integrin, alpha-9 integrin, alpha-D integrin, alpha-L integrin,alpha-M integrin, alpha-V integrin, alpha-X integrin, alpha-11bintegrin, alphaIELb integrin; Integrin-associated Molecules such as BetaIG-H3, Melusin, CD47, MEPE, CD151, Osteopontin, IBSP/Sialoprotein II,RAGE, IGSF8; Selectins such as E-Selectin, P-Selectin, L-Selectin; andLigands such as CD34, GlyCAM-1, MadCAM-1, PSGL-1, vitronectic,vitronectin receptor, fibronectin, vitronectin, collagen, laminin,ICAM-1, ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, and PECAM. Further markersof inflammation include cytokines such as IFN-α, IFN-β, IFN-ε, -κ, -π,and -ζ, IFN-ω, IFN-γ, IL29, IL28A and IL28B, IL-1, IL-1α and β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, and TCCR/WSX-1. Furthermarkers of inflammation include cytokine receptors such as Common betachain, IL-3 R alpha, IL-3 R beta, GM-CSF R, IL-5 R alpha, Common gammaChain/IL-2 R gamma, IL-2 R alpha, IL-9 R, IL-2 R beta, IL-4 R, IL-21 R,IL-15 R alpha, IL-7 R alpha/CD127, IL-1ra/IL-1F3, IL-1 R8, IL-1 RI, IL-1R9, IL-1 RII, IL-18 R alpha/IL-1 R5, IL-1 R3/IL-1 R AcP, IL-18 Rbeta/IL-1 R7, IL-1 R4/ST2 SIGIRR, IL-1 R6/IL-1 R rp2, IL-11 R alpha,IL-31 RA, CNTF R alpha, Leptin R, G-CSF R, LIF R alpha, IL-6 R, OSM Rbeta, IFN-alpha/beta R1, IFN-alpha/beta R2, IFN-gamma R1, IFN-gamma R2,IL10 R alpha, IL-10 R beta, IL-20 R alpha, IL-20 R beta, IL-22 R, IL-17R, IL-17 RD, IL-17 RC, IL-17B R, IL-13 R alpha 2, IL-23 R, IL-12 R beta1, IL-12 R beta 2, TCCR/WSX-1, and IL-13 R alpha 1. Further markers ofinflammation include chemokines such as CCL-1, CCL-2, CCL-3, CCL-4,CCL-5, CCL-6, CCL-7, CCL-8, CCL-9, CCL-10, CCL-11, CCL-12, CCL-13,CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-20, CCL-21, CCL-22,CCL-23, CCL-24, CCL-25, CCL-26, CCL-27, CCL-28, MCK-2, MIP-2, CINC-1,CINC-2, KC, CINC-3, LIX, ORO, Thymus Chemokine-1, CXCL-1, CXCL-2,CXCL-3, CXCL-4, CXCL-5, CXCL-6, CXCL-7, CXCL-8, CXCL-9, CXCL-10,CXCL-11, CXCL-12, CXCL-13, CXCL-14, CXCL-15, CXCL-16, CXCL-17, XCL1,XCL2, and Chemerin. Further markers of inflammation include chemokinereceptors such as CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7,CCR-8, CCR-9, CCR-10, CXCR3, CXCR6, CXCR4, CXCR1, CXCR5, CXCR2, ChemR23. Further markers of inflammation include Tumor necrosis factors(TNFs), such as TNF.alpha., 4-1BB Ligand/TNFSF9, LIGHT/TNFSF14,APRIL/TNFSF13, Lymphotoxin, BAFF/TNFSF13B, Lymphotoxin beta/TNFSF3, CD27Ligand/TNFSF7, OX40 Ligand/TNFSF4, CD30 Ligand/TNFSF8, TL1A/TNFSF15,CD40 Ligand/TNFSF5, TNF-alpha/TNFSF1A, EDA, TNF-beta/TNFSF1B, EDA-A2,TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18,and TWEAK/TNFSF12. Further markers of inflammation include TNFSuperfamily Receptors such as 4-1BB/TNFRSF9, NOF R/TNFRSF16, BAFFR/TNFRSF13C, Osteoprotgerin/TNFRSF11B, BCMA/TNFRSF17, OX40/TNFRSF4,CD27/TNFRSF7, RANK/TNFRSF11A, CD30/TNFRSF8, RELT/TNFRSF19L,CD40/TNFRSF5, TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF1A, DcTRAILR1/TNFRSF23, TNF RII/TNFRSF1B, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF10A,DR3/TNFRSF25, TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3TNFRSF10C, EDAR,TRAIL R4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAKR/TNFRSF12, HVEM/TNFRSF14, and XEDAR. Further markers of inflammationinclude TNF Superfamily Regulators such as FADD, TRAF-2, RIP1, TRAF-3,TRADD, TRAP-4, TRAF-I, and TRAF-6. Further markers of inflammationinclude acute-phase reactants and acute phase proteins. Further markersof inflammation include TGF-beta superfamily ligands such as Activins,Activin A, Activin B, Activin AB, Activin C, BMPs (Bone MorphogeneticProteins), BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4,BMP-10, BMP-5, BMP-15/GDP-9B, BMP-6, Decapentaplegic,Growth/Differentiation Factors (GDFs), GDF-1, GDF-8, GDF-3, GDF-9 GDF-5,GDF-11, GDF-6, GDF-15, GDF-7, GDNF Family Ligands, Artemin, Neurturin,GDNF, Persephin, TOP-beta, TGF-beta, TGF-beta 3, TGF-beta 1, TGF-beta 5,LAP (TGF-beta 1), Latent TGF-beta bp1, Latent TGF-beta 1, LatentTGF-beta bp2, TOP-beta 12, Latent TGF-beta bp4, TGF-beta 2, Lefty,MIS/AMH, Lefty-1, Nodal, Lefty-A, Activin RIA/ALK-2, GFR alpha-1/GDNF Ralpha-1, Activin RIB/ALK-4, GFR alpha-2/GDNF R alpha-2, Activin RIIA,GFR alpha-3/GDNF R alpha-3, Activin RIIB, GFR alpha-4/GDNF R alpha-4,ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TOP-beta RI/ALK-5,BMPR-IB/ALK-6, TGF-beta RII, BMPR-II, TGF-beta RIIb, Endoglin/CD105, andTGF-beta RIII. Further markers of inflammation include TGF-betasuperfamily Modulators such as Amnionless, NCAM-1/CD56, BAMBI/NMA,Noggin, BMP-1/PCP, NOMO, Caronte, PRDC, Cerberus 1, SKI, Chordin, Smad1,Chordin-Like 1, Smad2, Chordin-Like 2, Smad3, COCO, Smad4, CRIM1, Smad5,Cripto, Smad7, Crossveinless-2, Smad8, Cryptic, SOST, DAN, LatentTOP-beta bp1, Decorin, Latent TGF-beta bp2, FLRG, Latent TOP-beta bp4,Follistatin, TMEFF1/Tomoregulin-1, Follistatin-like 1, TMEFF2,GASP-1/WFIKKNRP, TSG, GASP-2/WFIKKN, TSK, Gremlin, and Vasorin. Furthermarkers of inflammation include EGP Ligands such as Amphiregulin, LRIG3,Betacellulin, Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen,TGF-alpha, Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, and LRIG1.Further markers of inflammation include EGF R/ErbB Receptor Family, suchas EGF R, ErbB3, ErbB2, and ErbB4. Further markers of inflammationinclude Fibrinogen. Further markers of inflammation include SAA. Furthermarkers of inflammation include glial markers, such asalpha.1-antitrypsin, C-reactive protein (CRP), .alpha.2-macroglobulin,glial fibrillary acidic protein (GFAP), Mac-1, and F4/80. Furthermarkers of inflammation include myeloperoxidase. Further markers ofinflammation include Complement markers such as C3d, C1q, C5, C4d, C4bp,and C5a-C9. Further markers of inflammation include Majorhistocompatibility complex (MHC) glycoproteins, such as HLA-DR andHLA-A,D,C. Further markers of inflammation include Microglial markers,such as CR3 receptor, MHC I, MHC H, CD 31, CD11a, CD11b, CD11c, CD68,CD45RO, CD45RD, CD18, CD59, CR4, CD45, CD64, and CD44. Further markersof inflammation include alpha.2 macroglobulin receptor, Fibroblastgrowth factor, Fc gamma RI, Fc gamma RII, CD8, LCA (CD45), CD18 ( ),CD59, Apo J, clusterin, type 2 plasminogen activator inhibitor, CD44,Macrophage colony stimulating factor receptor, MRP14, 27E10,4-hydroxynonenal-protein conjugates, I.kappa.B, NF.kappa.B, cPLA.sub.2,COX-2, Matrix metalloproteinases, Membrane lipid peroxidation, andATPase activity. HSPC228, EMP1, CDC42, TLE3, SPRY2, p40BBP, HSPC060 andNAB2, or a down-regulation of HSPA1A, HSPA1B, MAPRE2 and OAS1expression, TACE/ADAM17, alpha-1-Acid Glycoprotein, Angiopoietin-1, MIF,Angiopoietin-2, CD14, beta-Defensin 2, MMP-2, ECF-L/CHI3L3, MMP-7, EGF,MMP-9, EMAP-II, MSP, EN-RAGE, Nitric Oxide, Endothelin-1,Osteoactivin/GPNMB, FPR1, PDGF, FPRL1, Pentraxin 3/TSG-14, FPRL2, Gas6,PLUNC, GM-CSF, RAGE, S100A10, S100A8, S100A9, HIF-1 alpha, Substance P,TFPI, TOP-beta 1, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLR4, LBP, TREM-1,Leukotriene A4, Hydrolase TSG-6, Lipocalin-1, uPA, M-CSF, and VEGF.

Miscellaneous Markers

Oncology markers that can be used in methods and compositions of theinvention include EGF, TNF-alpha, PSA, VEGF, TGF-beta1, FGFb, TRAIL, andTNF-RI (p55).

Markers of endocrine function that can be used in methods andcompositions of the invention include 17 beta-estradiol (E2), DHEA,ACTH, gastrin, and growth hormone (hGH).

Markers of autoimmunity that can be used in methods and compositions ofthe invention include GM-CSF, C-Reactive Protein, and G-CSF.

Markers of thyroid function that can be used in methods and compositionsof the invention include cyclicAMP, calcitonin, and parathyroid hormone.

Cardiovascular markers that can be used in methods and compositions ofthe invention include cardiac troponin I, cardiac troponin T,B-natriuretic peptide, NT-proBNP, C-ractive Protein HS, andbeta-thromboglobulin.

Markers of diabetes that can be used in methods and compositions of theinvention include C-peptide and leptin.

Markers of infectious disease that can be used in methods andcompositions of the invention include IFN-gamma and IFN-alpha.

Markers of metabolism that can be used in methods and compositions ofthe invention include Bio-intact PTH (1-84) and PTH.

Markers of Biological States

Markers can indicate the presence of a particular phenotypic state ofinterest. Examples of phenotypic states include, phenotypes resultingfrom an altered environment, drug treatment, genetic manipulations ormutations, injury, change in diet, aging, or any other characteristic(s)of a single organism or a class or subclass of organisms.

In some embodiments, a phenotypic state of interest is a clinicallydiagnosed disease state. Such disease states include, for example,cancer, cardiovascular disease, inflammatory disease, autoimmunedisease, neurological disease, infectious disease and pregnancy relateddisorders. Alternatively, states of health can be detected usingmarkers.

Cancer phenotypes are included in some aspects of the invention.Examples of cancer herein include, but are not limited to: breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gallbladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell, lung tumor, non-small cell lungcarcinoma gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas,intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, epidermoid carcinomas, and othercarcinomas and sarcomas.

Cardiovascular disease can be included in other applications of theinvention. Examples of cardiovascular disease include, but are notlimited to, congestive heart failure, high blood pressure, arrhythmias,atherosclerosis, cholesterol, Wolff-Parkinson-White Syndrome, long QTsyndrome, angina pectoris, tachycardia, bradycardia, atrialfibrillation, ventricular fibrillation, myocardial ischemia, myocardialinfarction, cardiac tamponade, myocarditis, pericarditis, arrhythmogenicright ventricular dysplasia, hypertrophic cardiomyopathy, Williamssyndrome, heart valve diseases, endocarditis, bacterial disease,pulmonary atresia, aortic valve stenosis, Raynaud's disease, cholesterolembolism, Wallenberg syndrome, Hippel-Lindau disease, andtelangiectasis.

Inflammatory disease and autoimmune disease can be included in otherembodiments of the invention. Examples of inflammatory disease andautoimmune disease include, but are not limited to, rheumatoidarthritis, non-specific arthritis, inflammatory disease of the larynx,inflammatory bowel disorder, psoriasis, hypothyroidism (e.g., Hashimotothyroidism), colitis, Type 1 diabetes, pelvic inflammatory disease,inflammatory disease of the central nervous system, temporal arteritis,polymyalgia rheumatica, ankylosing spondylitis, polyarteritis nodosa,Reiter's syndrome, scleroderma, systemis lupus and erythematosus.

The methods and compositions of the invention can also providelaboratory information about markers of infectious disease includingmarkers of Adenovirus, Bordella pertussis, Chlamydia pneumoiea,Chlamydia trachomatis, Cholera Toxin, Cholera Toxin β, Campylobacterjejuni, Cytomegalovirus, Diptheria Toxin, Epstein-Barr NA, Epstein-BarrEA, Epstein-Barr VCA, Helicobacter Pylori, Hepatitis B virus (HBV) Core,Hepatitis B virus (HBV) Envelope, Hepatitis B virus (HBV) Surface (Ay),Hepatitis C virus (HCV) Core, Hepatitis C virus (HCV) NS3, Hepatitis Cvirus (HCV) NS4, Hepatitis C virus (HCV) NS5, Hepatitis A, Hepatitis D,Hepatitis E virus (HEV) orf2 3KD, Hepatitis E virus (HEV) orf2 6KD,Hepatitis E virus (HEV) orf3 3KD, Human immunodeficiency virus (HIV)-1p24, Human immunodeficiency virus (HIV)-1 gp41, Human immunodeficiencyvirus (HIV)-1 gp120, Human papilloma virus (HPV), Herpes simplex virusHSV-1/2, Herpes simplex virus HSV-1 gD, Herpes simplex virus HSV-2 gG,Human T-cell leukemia virus (HTLV)-1/2, Influenza A, Influenza A H3N2,Influenza B, Leishmania donovani, Lyme disease, Mumps, M. pneumoniae, M.tuberculosis, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, PolioVirus, Respiratory syncytial virus (RSV), Rubella, Rubeola, StreptolysinO, Tetanus Toxin, T. pallidum 15kd, T. pallidum p47, T. cruzi,Toxoplasma, and Varicella Zoster.

IV. Labels

In some embodiments, the invention provides methods and compositionsthat include labels for the highly sensitive detection and quantitationof molecules, e.g., of markers.

One skilled in the art will recognize that many strategies can be usedfor labeling target molecules to enable their detection ordiscrimination in a mixture of particles. The labels can be attached byany known means, including methods that utilize non-specific or specificinteractions of label and target. Labels can provide a detectable signalor affect the mobility of the particle in an electric field. Labelingcan be accomplished directly or through binding partners.

In some embodiments, the label comprises a binding partner to themolecule of interest, where the binding partner is attached to afluorescent moiety. The compositions and methods of the invention canuse highly fluorescent moieties. Moieties suitable for the compositionsand methods of the invention are described in more detail below.

In some embodiments, the invention provides a label for detecting abiological molecule comprising a binding partner for the biologicalmolecule that is attached to a fluorescent moiety, wherein thefluorescent moiety is capable of emitting at least about 200 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, wherein the laser is focused on a spot not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the moiety comprises a plurality offluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or3 to 10 fluorescent entities. In some embodiments, the moiety comprisesabout 2 to 4 fluorescent entities. In some embodiments, the biologicalmolecule is a protein or a small molecule. In some embodiments, thebiological molecule is a protein. The fluorescent entities can befluorescent dye molecules. In some embodiments, the fluorescent dyemolecules comprise at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. In someembodiments, the dye molecules are Alexa Fluor molecules selected fromthe group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor647, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the dyemolecules are Alexa Fluor molecules selected from the group consistingof Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700.In some embodiments, the dye molecules are Alexa Fluor 647 dyemolecules. In some embodiments, the dye molecules comprise a first typeand a second type of dye molecules, e.g., two different Alexa Fluormolecules, e.g., where the first type and second type of dye moleculeshave different emission spectra. The ratio of the number of first typeto second type of dye molecule can be, e.g., 4 to 1, 3 to 1, 2 to 1, 1to 1, 1 to 2, 1 to 3 or 1 to 4. The binding partner can be, e.g., anantibody.

In some embodiments, the invention provides a label for the detection ofa marker, wherein the label comprises a binding partner for the markerand a fluorescent moiety, wherein the fluorescent moiety is capable ofemitting at least about 200 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, wherein the laser isfocused on a spot not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the fluorescent moiety comprises a fluorescent molecule. In someembodiments, the fluorescent moiety comprises a plurality of fluorescentmolecules, e.g., about 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8,or 3 to 6 fluorescent molecules. In some embodiments, the labelcomprises about 2 to 4 fluorescent molecules. In some embodiments, thefluorescent dye molecules comprise at least one substituted indoliumring system in which the substituent on the 3-carbon of the indoliumring contains a chemically reactive group or a conjugated substance. Insome embodiments, the fluorescent molecules are selected from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, AlexaFluor 680 or Alexa Fluor 700. In some embodiments, the fluorescentmolecules are selected from the group consisting of Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700. In someembodiments, the fluorescent molecules are Alexa Fluor 647 molecules. Insome embodiments, the binding partner comprises an antibody. In someembodiments, the antibody is a monoclonal antibody. In otherembodiments, the antibody is a polyclonal antibody.

The antibody can be specific to any suitable marker. In someembodiments, the antibody is specific to a marker that is selected fromthe group consisting of cytokines, growth factors, oncology markers,markers of inflammation, endocrine markers, autoimmune markers, thyroidmarkers, cardiovascular markers, markers of diabetes, markers ofinfectious disease, neurological markers, respiratory markers,gastrointestinal markers, musculoskeletal markers, dermatologicaldisorders, and metabolic markers.

In some embodiments, the antibody is specific to a marker that is acytokine. In some embodiments, the cytokine is selected from the groupconsisting of BDNF, CREB pS133, CREB Total, DR-5, EGF, ENA-78, Eotaxin,Fatty Acid Binding Protein, FGF-basic, granulocyte colony-stimulatingfactor (G-CSF), GCP-2, Granulocyte-macrophage Colony-stimulating FactorGM-CSF (GM-CSF), growth-related oncogene-keratinocytes (GRO-KC), HGF,ICAM-1, IFN-alpha, IFN-gamma, the interleukins IL-10, IL-11, IL-12,IL-12 p40, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-16, IL-17, IL-18,IL-1alpha, IL-1beta, IL-1ra, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5,II-6, IL-7, IL-8, IL-9, interferon-inducible protein (10 IP-10),JE/MCP-1, keratinocytes (KC), KC/GROa, LIP, Lymphotacin, M-CSF, monocytechemoattractant protein-1 (MCP-1), MCP-1(MCAF), MCP-3, MCP-5, MDC, MIG,macrophage inflammatory (MIP-1 alpha), MIP-1 beta, MIP-1 gamma, MIP-2,MIP-3 beta, OSM, PDGF-BB, regulated upon activation-normal Tcell-expressed and secreted (RANTES), Rb (pT821), Rb (total), RbpSpT249/252, Tau (pS214), Tau (pS396), Tau (total), Tissue Factor, tumornecrosis factor-alpha (TNF-alpha), TNF-beta, TNF-RI, TNF-RII, VCAM-1,and VEGF.

In some embodiments, the cytokine is selected from the group consistingof IL-12 p70, IL-10, IL-1 alpha, IL-3, IL-12 p40, IL-1ra, IL-12, IL-6,IL-4, IL-18, IL-10, IL-5, Eotaxin, IL-16, MIG, IL-8, IL-17, IL-7, IL-15,IL-13, IL-2R (soluble), IL-2, LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1 andMCP-1.

In some embodiments, the antibody is specific to a marker that is agrowth factor (GF). In some embodiments, the antibody is specific to amarker that is a growth factor that is TGF-beta. In some embodiments,the growth factor is a GF ligand such as Amphiregulin, LRIG3,Betacellulin, Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen,TGF-alpha, Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1; EGFR/ErbB Receptor Family such as EGF R, ErbB3, ErbB2, ErbB4; FGF Familysuch as FGF Ligands, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3,FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21,FGF-9, FGF-22, FGF-10, FGF-23, FGF-11, KGF/FGF-7, FGF Receptors FGFR1-4, FGF R3, FGF R, FGF R4, FGF R2, FGF R5, FGF Regulators FGF-BP; theHedgehog Family Desert Hedgehog, Sonic Hedgehog, Indian Hedgehog;Hedgehog Related Molecules & Regulators BOC, GLI-3, CDO, GSK-3alpha/beta, DISP1, GSK-3 alpha, Gas1, GSK-3 beta, GLI-1, Hip, GLI-2; theIGF Family IGF ligands IGF-I, IGF-II, IGF-I Receptor (CD221) IGF-I R,and IGF Binding Protein (IGFBP) Family ALS, IGFBP-5, CTGF/CCN2, IGFBP-6,Cyr61/CCN1, IGFBP-L1, Endocan, IGFBP-rp1/IGFBP-7, IGFBP-1, IGFBP-rP10,IGFBP-2, NOV/CCN3, IGFBP-3, WISP-1/CCN4, IGFBP-4; Receptor TyrosineKinases Ax1, FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk,IGF-I R, EGF, R IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2,M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha,EphA7, PDGF R beta, EphA8, Ret, EphB1, RTK-like Orphan Receptor 1/ROR1,EphB2, RTK-like Orphan Receptor 2/ROR2, EphB3, SCF R/c-kit, EphB4,Tie-1, EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, POP, RI-4VEGF R, FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/KDR/Flk-1, FGF R3, VEGFR3/Flt-4; Proteoglycans & Regulators Proteoglycans Aggrecan, Mimecan,Agrin, NG2/MCSP, Biglycan, Osteoadherin, Decorin, Podocan, DSPG3,delta-Sarcoglycan, Endocan, Syndecan-1/CD138, Endoglycan, Syndecan-2,Endorepellin/Perlecan, Syndecan-3, Glypican 2, Syndecan-4, Glypican 3,Testican 1/SPOCK1, Glypican 5, Testican 2/SPOCK2, Glypican 6, Testican3/SPOCK3, Lumican, Versican, Proteoglycan Regulators, ArylsulfataseA/ARSA, Glucosamine (N-acetyl)-6-Sulfatase/GNS, Exostosin-like 2/EXTL2,HS6ST2, Exostosin-like 3/EXTL3, Iduronate 2-Sulfatase/IDS, GalNAc4S-6ST;SCF, Fit-3 Ligand & M-CSF Flt-3, M-CSF R, Flt-3 Ligand, SCF, M-CSF, SCFR/c-kit; TOP-beta Superfamily (same as listed for inflammatory markers);VEGF/PDGF Family Neuropilin-1, PlGF, Neuropilin-2, PlGF-2, PDGF, VEGF,PDGF R alpha, VEGF-B, PDGF R beta, VEGF-C, PDGF-A, VEGF-D, PDGF-AB, VEGFR, PDGF-B, VEGF R1/Flt-1, PDGF-C, VEGF R2/KDR/Flk-1, PDGF-D, VEGFR3/Flt-4; Wnt-related Molecules Dickkopf Proteins & Wnt InhibitorsDkk-1, Dkk-4, Dkk-2, Soggy-1, Dkk-3, WIF-1 Frizzled & Related ProteinsFrizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1,Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7,MFRP Wnt Ligands Wnt-1, Wnt-8a, Wnt-2b, Wnt-8b, Wnt-3a, Wnt-9a, Wnt-4,Wnt-9b, Wnt-5a, Wnt-10a, Wnt-5b, Wnt-10b, Wnt-7a, Wnt-11, Wnt-7b; OtherWnt-related Molecules APC, Kremen-2, Axin-1, LRP-1, beta-Catenin, LRP-6,Dishevelled-1, Norrin, Dishevelled-3, PKC beta 1, Glypican 3, Pygopus-1,Glypican 5, Pygopus-2, GSK-3 alpha/beta, R-Spondin 1, GSK-3 alpha,R-Spondin 2, GSK-3 beta, R-Spondin 3, ICAT, RTK-like Orphan Receptor1/ROR1, Kremen-1, RTK-like Orphan Receptor 2/ROR, and Other GrowthFactors CTGF/CCN2, beta-NOF, Cyr61/CCN1, Norrin, DANCE, NOV/CCN3,EG-VEGF/PK1, Osteocrin, Hepassocin, PD-ECGF, HGF, Progranulin, LECT2,Thrombopoietin, LEDGF, or WISP-1/CCN4.

In some embodiments, the antibody is specific to a marker that is amarker for cancer (oncology marker). In some embodiments, the antibodyis specific to a marker that is a marker for cancer that is EGF. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is TNF-alpha. In some embodiments, the antibody is specificto a marker that is a marker for cancer that is PSA. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is VEGF. In some embodiments, the antibody is specific to amarker that is a marker for cancer that is TOP-beta. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is FGFb. In some embodiments, the antibody is specific to amarker that is a marker for cancer that is TRAIL. In some embodiments,the antibody is specific to a marker that is a marker for cancer that isTNF-RI (p55).

In further embodiments, the antibody is specific to a marker for cancerthat is alpha-Fetoprotein. In some embodiments, the antibody is specificto a marker for cancer that is ER beta/NR3A2. In some embodiments, theantibody is specific to a marker for cancer that is ErbB2. In someembodiments, the antibody is specific to a marker for cancer that isKallikrein 3/PSA. In some embodiments, the antibody is specific to amarker for cancer that is ER alpha/NR3A1. In some embodiments, theantibody is specific to a marker for cancer that is ProgesteroneR/NR3C3. In some embodiments, the antibody is specific to a marker forcancer that is A33. In some embodiments, the antibody is specific to amarker for cancer that is MIA. In some embodiments, the antibody isspecific to a marker for cancer that is Aurora A. In some embodiments,the antibody is specific to a marker for cancer that is MMP-2. In someembodiments, the antibody is specific to a marker for cancer that isBcl-2. In some embodiments, the antibody is specific to a marker forcancer that is MMP-3. In some embodiments, the antibody is specific to amarker for cancer that is Cadherin-13. In some embodiments, the antibodyis specific to a marker for cancer that is MMP-9. In some embodiments,the antibody is specific to a marker for cancer that is E-Cadherin. Insome embodiments, the antibody is specific to a marker for cancer thatis NEK2. In some embodiments, the antibody is specific to a marker forcancer that is Carbonic Anhydrase IX In some embodiments, the antibodyis specific to a marker for cancer that is Nestin. In some embodiments,the antibody is specific to a marker for cancer that is beta-Catenin. Insome embodiments, the antibody is specific to a marker for cancer thatis NG2/MCSP. In some embodiments, the antibody is specific to a markerfor cancer that is Cathepsin D. In some embodiments, the antibody isspecific to a marker for cancer that is Osteopontin. In someembodiments, the antibody is specific to a marker for cancer that isCD44. In some embodiments, the antibody is specific to a marker forcancer that is p21/CIP1/CDKN1A. In some embodiments, the antibody isspecific to a marker for cancer that is CEACAM-6. In some embodiments,the antibody is specific to a marker for cancer that is p27/Kip1. Insome embodiments, the antibody is specific to a marker for cancer thatis Comulin. In some embodiments, the antibody is specific to a markerfor cancer that is p53. In some embodiments, the antibody is specific toa marker for cancer that is DPPA4. In some embodiments, the antibody isspecific to a marker for cancer that is Prolactin. In some embodiments,the antibody is specific to a marker for cancer that is ECM-1. In someembodiments, the antibody is specific to a marker for cancer that isPSP94. In some embodiments, the antibody is specific to a marker forcancer that is EGF. In some embodiments, the antibody is specific to amarker for cancer that is S100B. In some embodiments, the antibody isspecific to a marker for cancer that is EGF R. In some embodiments, theantibody is specific to a marker for cancer that is S100P. In someembodiments, the antibody is specific to a marker for cancer that isEMMPRIN/CD 147. In some embodiments, the antibody is specific to amarker for cancer that is SCF R/c-kit. In some embodiments, the antibodyis specific to a marker for cancer that is Fibroblast Activation Proteinalpha/PAP. In some embodiments, the antibody is specific to a marker forcancer that is Serpin E1/PAI-1. In some embodiments, the antibody isspecific to a marker for cancer that is FGF acidic. In some embodiments,the antibody is specific to a marker for cancer that is Serum AmyloidA4. In some embodiments, the antibody is specific to a marker for cancerthat is FGF basic. In some embodiments, the antibody is specific to amarker for cancer that is Survivin. In some embodiments, the antibody isspecific to a marker for cancer that is Galectin-3. In some embodiments,the antibody is specific to a marker for cancer that is TEM8. In someembodiments, the antibody is specific to a marker for cancer that isGlypican 3. In some embodiments, the antibody is specific to a markerfor cancer that is TIMP-1. In some embodiments, the antibody is specificto a marker for cancer that is HIN-1/Secretoglobulin 3A1. In someembodiments, the antibody is specific to a marker for cancer that isTIMP-2. In some embodiments, the antibody is specific to a marker forcancer that is IGF-I. In some embodiments, the antibody is specific to amarker for cancer that is TIMP-3. In some embodiments, the antibody isspecific to a marker for cancer that is IGFBP-3. In some embodiments,the antibody is specific to a marker for cancer that is TIMP-4. In someembodiments, the antibody is specific to a marker for cancer that isIL-6. In some embodiments, the antibody is specific to a marker forcancer that is TNF-alpha/TNFSF1A. In some embodiments, the antibody isspecific to a marker for cancer that is Kallikrein 6/Neurosin. In someembodiments, the antibody is specific to a marker for cancer that isTRAP-4. In some embodiments, the antibody is specific to a marker forcancer that is M-CSF. In some embodiments, the antibody is specific to amarker for cancer that is uPA. In some embodiments, the antibody isspecific to a marker for cancer that is Matriptase/ST14. In someembodiments, the antibody is specific to a marker for cancer that isuPAR. In some embodiments, the antibody is specific to a marker forcancer that is Mesothelin. In some embodiments, the antibody is specificto a marker for cancer that is VCAM-1. In some embodiments, the antibodyis specific to a marker for cancer that is Methionine Aminopeptidase. Insome embodiments, the antibody is specific to a marker for cancer thatis VEGF. In some embodiments, the antibody is specific to a marker forcancer that is Methionine Aminopeptidase 2.

In some embodiments, the antibody is specific to a marker that is amarker for inflammation. In some embodiments, the antibody is specificto a marker that is a marker for inflammation that is ICAM-1. In someembodiments, the antibody is specific to a marker that is a marker forinflammation that is RANTES. In some embodiments, the antibody isspecific to a marker that is a marker for inflammation that is MIP-2. Insome embodiments, the antibody is specific to a marker that is a markerfor inflammation that is MIP-1 beta. In some embodiments, the antibodyis specific to a marker that is a marker for inflammation that is MIP-1alpha. In some embodiments, the antibody is specific to a marker that isa marker for inflammation that is MMP-3.

In some embodiments, the antibody is specific to a marker that is amarker for endocrine function. In some embodiments, the antibody isspecific to a marker that is a marker for endocrine function that is 17beta-estradiol (E2). In some embodiments, the antibody is specific to amarker that is a marker for endocrine function that is DHEA. In someembodiments, the antibody is specific to a marker that is a marker forendocrine function that is ACTH. In some embodiments, the antibody isspecific to a marker that is a marker for endocrine function that isgastrin. In some embodiments, the antibody is specific to a marker thatis a marker for endocrine function that is growth hormone.

In some embodiments, the antibody is specific to a marker that is amarker for autoimmune disease. In some embodiments, the antibody isspecific to a marker that is a marker for autoimmune disease that isGM-CSF. In some embodiments, the antibody is specific to a marker thatis a marker for autoimmune disease that is C-reactive protein (CRP). Insome embodiments, the antibody is specific to a marker that is a markerfor autoimmune disease that is G-CSF.

In some embodiments, the antibody is specific to a marker for thyroidfunction. In some embodiments, the antibody is specific to a marker forthyroid function that is cyclic AMP. In some embodiments, the antibodyis specific to a marker for thyroid function. In some embodiments, theantibody is specific to a marker for thyroid function that iscalcitonin. In some embodiments, the antibody is specific to a markerfor thyroid function. In some embodiments, the antibody is specific to amarker for thyroid function that is parathyroid hormone.

In some embodiments, the antibody is specific to a marker forcardiovascular function. In some embodiments, the antibody is specificto a marker for cardiovascular function that is B-natriuretic peptide.In some embodiments, the antibody is specific to a marker forcardiovascular function that is NT-proBNP. In some embodiments, theantibody is specific to a marker for cardiovascular function that isC-reactive protein, HS. In some embodiments, the antibody is specific toa marker for cardiovascular function that is beta-thromboglobulin. Insome embodiments, the antibody is specific to a marker forcardiovascular function that is a cardiac troponin. In some embodiments,the antibody is specific to a marker for cardiovascular function that iscardiac troponin I. In some embodiments, the antibody is specific to amarker for cardiovascular function that is cardiac troponin T.

In some embodiments, the antibody is specific to a marker for diabetes.In some embodiments, the antibody is specific to a marker for diabetesthat is C-peptide. In some embodiments, the antibody is specific to amarker for diabetes that is leptin.

In some embodiments, the antibody is specific to a marker for infectiousdisease. In some embodiments, the antibody is specific to a marker forinfectious disease that is IFN gamma. In some embodiments, the antibodyis specific to a marker for infectious disease that is IFN alpha. Insome embodiments, the antibody is specific to a marker for infectiousdisease that is TREM-1.

In some embodiments, the antibody is specific to a marker formetabolism. In some embodiments, the antibody is specific to a markerfor metabolism that is bio-intact PTH (1-84). In some embodiments, theantibody is specific to a marker for metabolism that is PTH.

In some embodiments, the antibody is specific to a marker that is IL-1beta. In some embodiments, the antibody is specific to a marker that isTNP-alpha. In some embodiments, the antibody is specific to a markerthat is IL-6. In some embodiments, the antibody is specific to a markerthat is TnI (cardiac troponin I). In some embodiments, the antibody isspecific to a marker that is IL-8.

In some embodiments, the antibody is specific to a marker that is Abeta40. In some embodiments, the antibody is specific to a marker that isAbeta 42. In some embodiments, the antibody is specific to a marker thatis cAMP. In some embodiments, the antibody is specific to a marker thatis FAS Ligand. In some embodiments, the antibody is specific to a markerthat is FGF-basic. In some embodiments, the antibody is specific to amarker that is GM-CSF. In some embodiments, the antibody is specific toa marker that is IFN-alpha. In some embodiments, the antibody isspecific to a marker that is IFN-gamma. In some embodiments, theantibody is specific to a marker that is IL-1a. In some embodiments, theantibody is specific to a marker that is IL-2. In some embodiments, theantibody is specific to a marker that is IL-4. In some embodiments, theantibody is specific to a marker that is IL-5. In some embodiments, theantibody is specific to a marker that is IL-7. In some embodiments, theantibody is specific to a marker that is IL-12. In some embodiments, theantibody is specific to a marker that is IL-13. In some embodiments, theantibody is specific to a marker that is IL-17. In some embodiments, theantibody is specific to a marker that is MCP-1. In some embodiments, theantibody is specific to a marker that is MIP-1a. In some embodiments,the antibody is specific to a marker that is RANTES. In someembodiments, the antibody is specific to a marker that is VEGF.

In some embodiments, the antibody is specific to a marker that is ACE.In some embodiments, the antibody is specific to a marker that isactivin A. In some embodiments, the antibody is specific to a markerthat is adiponectin. In some embodiments, the antibody is specific to amarker that is adipsin. In some embodiments, the antibody is specific toa marker that is AgRP. In some embodiments, the antibody is specific toa marker that is AKT1. In some embodiments, the antibody is specific toa marker that is albumin. In some embodiments, the antibody is specificto a marker that is betacellulin. In some embodiments, the antibody isspecific to a marker that is bombesin. In some embodiments, the antibodyis specific to a marker that is CD14. In some embodiments, the antibodyis specific to a marker that is CD-26. In some embodiments, the antibodyis specific to a marker that is CD-38. In some embodiments, the antibodyis specific to a marker that is CD-40L. In some embodiments, theantibody is specific to a marker that is CD-40s. In some embodiments,the antibody is specific to a marker that is CDK5. In some embodiments,the antibody is specific to a marker that is Complement C3. In someembodiments, the antibody is specific to a marker that is Complement C4.In some embodiments, the antibody is specific to a marker that isC-peptide. In some embodiments, the antibody is specific to a markerthat is CRP. In some embodiments, the antibody is specific to a markerthat is EGF. In some embodiments, the antibody is specific to a markerthat is E-selectin. In some embodiments, the antibody is specific to amarker that is FAS. In some embodiments, the antibody is specific to amarker that is FASLG. In some embodiments, the antibody is specific to amarker that is Fetuin A. In some embodiments, the antibody is specificto a marker that is fibrinogen. In some embodiments, the antibody isspecific to a marker that is ghrelin. In some embodiments, the antibodyis specific to a marker that is glucagon. In some embodiments, theantibody is specific to a marker that is growth hormone. In someembodiments, the antibody is specific to a marker that is haptoglobulin.In some embodiments, the antibody is specific to a marker that ishepatocyte growth factor. In some embodiments, the antibody is specificto a marker that is HGF. In some embodiments, the antibody is specificto a marker that is ICAM1. In some embodiments, the antibody is specificto a marker that is IFNG. In some embodiments, the antibody is specificto a marker that is IGF1. In some embodiments, the antibody is specificto a marker that is IL-1RA. In some embodiments, the antibody isspecific to a marker that is Il-6sr. In some embodiments, the antibodyis specific to a marker that is IL-8. In some embodiments, the antibodyis specific to a marker that is IL-10. In some embodiments, the antibodyis specific to a marker that is IL-18. In some embodiments, the antibodyis specific to a marker that is ILGFBP1. In some embodiments, theantibody is specific to a marker that is ILGFBP3. In some embodiments,the antibody is specific to a marker that is insulin-like growthfactor 1. In some embodiments, the antibody is specific to a marker thatis LEP. In some embodiments, the antibody is specific to a market thatis M-CSF. In some embodiments, the antibody is specific to a marker thatis MMP2. In some embodiments, the antibody is specific to a marker thatis MMP9. In some embodiments, the antibody is specific to a marker thatis NOF. In some embodiments, the antibody is specific to a marker thatis PAI-1. In some embodiments, the antibody is specific to a marker thatis RAGE In some embodiments, the antibody is specific to a marker thatis RSP4. In some embodiments, the antibody is specific to a marker thatis resistin. In some embodiments, the antibody is specific to a markerthat is sex hormone binding globulin. In some embodiments, the antibodyis specific to a marker that is SOCX3. In some embodiments, the antibodyis specific to a marker that is TOP beta. In some embodiments, theantibody is specific to a marker that is thromboplastin. In someembodiments, the antibody is specific to a marker that is TNF R1. Insome embodiments, the antibody is specific to a marker that is VCAM-1.In some embodiments, the antibody is specific to a marker that is VWF.In some embodiments, the antibody is specific to a marker that is TSH.In some embodiments, the antibody is specific to a marker that isEPITOME.

In some embodiments, the antibody is specific to a marker correspondingto the molecule of interest. In some embodiments, the antibody isspecific to a marker that is cardiac troponin I. In some embodiments,the antibody is specific to a marker that is TREM-1. In someembodiments, the antibody is specific to a marker that is IL-6. In someembodiments, the antibody is specific to a marker that is IL-8. In someembodiments, the antibody is specific to a marker that is LeukotrieneT4. In some embodiments, the antibody is specific to a marker that isAkt1. In some embodiments, the antibody is specific to a marker that isTGF-beta. In some embodiments, the antibody is specific to a marker thatis Fas ligand.

A. Binding Partners

Any suitable binding partner with the requisite specificity for the formof molecule, e.g., a marker, to be detected can be used. If themolecule, e.g., a marker, has several different forms, variousspecificities of binding partners are possible. Suitable bindingpartners are known in the art and include antibodies, aptamers, lectins,and receptors. A useful and versatile type of binding partner is anantibody.

1. Antibodies

In some embodiments, the binding partner is an antibody specific for amolecule to be detected. The term “antibody,” as used herein, is a broadterm and is used in its ordinary sense, including, without limitation,to refer to naturally occurring antibodies as well as non-naturallyoccurring antibodies, including, for example, single chain antibodies,chimeric, bifunctional and humanized antibodies, as well asantigen-binding fragments thereof. It will be appreciated that thechoice of epitope or region of the molecule to which the antibody israised will determine its specificity, e.g., for various forms of themolecule, if present, or for total (e.g., all, or substantially all, ofthe molecule).

Methods for producing antibodies are well-established. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments that mimicantibodies can be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebacck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). Monoclonal and polyclonal antibodies to molecules, e.g.,proteins, and markers also commercially available (R and D Systems,Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc.,Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA;Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049USA; BiosPacific, Emeryville, Calif.).

In some embodiments, the antibody is a polyclonal antibody. In otherembodiments, the antibody is a monoclonal antibody.

Capture binding partners and detection binding partner pairs, e.g.,capture and detection antibody pairs, can be used in embodiments of theinvention. Thus, in some embodiments, a heterogeneous assay protocol isused in which, typically, two binding partners, e.g., two antibodies,are used. One binding partner is a capture partner, usually immobilizedon a solid support, and the other binding partner is a detection bindingpartner, typically with a detectable label attached. Such antibody pairsare available from the sources described above, e.g., BiosPacific,Emeryville, Calif. Antibody pairs can also be designed and prepared bymethods well-known in the art. Compositions of the invention includeantibody pairs wherein one member of the antibody pair is a label asdescribed herein, and the other member is a capture antibody.

In some embodiments it is useful to use an antibody that cross-reactswith a variety of species, either as a capture antibody, a detectionantibody, or both. Such embodiments include the measurement of drugtoxicity by determining, e.g., release of cardiac troponin into theblood as a marker of cardiac damage. A cross-reacting antibody allowsstudies of toxicity to be done in one species, e.g. a non-human species,and direct transfer of the results to studies or clinical observationsof another species, e.g., humans, using the same antibody or antibodypair in the reagents of the assays, thus decreasing variability betweenassays. Thus, in some embodiments, one or more of the antibodies for useas a binding partner to the marker of the molecule of interest, e.g.,cardiac troponin, such as cardiac troponin I, can be a cross-reactingantibody. In some embodiments, the antibody cross-reacts with themarker, e.g. cardiac troponin, from at least two species selected fromthe group consisting of human, monkey, dog, and mouse. In someembodiments, the antibody cross-reacts with the marker, e.g., cardiactroponin, from the entire group consisting of human, monkey, dog, andmouse.

B. Fluorescent Moieties

In some embodiments of labels used in the invention, the bindingpartner, e.g. an antibody, is attached to a fluorescent moiety. Thefluorescence of the moiety can be sufficient to allow detection in asingle molecule detector, such as the single molecule detectorsdescribed herein.

A “fluorescent moiety,” as that term is used herein, includes one ormore fluorescent entities whose total fluorescence is such that themoiety can be detected in the single molecule detectors describedherein. Thus, a fluorescent moiety can comprise a single entity (e.g., aQuantum Dot or fluorescent molecule) or a plurality of entities (e.g., aplurality of fluorescent molecules). It will be appreciated that when“moiety,” as that term is used herein, refers to a group of fluorescententities, e.g., a plurality of fluorescent dye molecules, eachindividual entity can be attached to the binding partner separately orthe entities can be attached together, as long as the entities as agroup provide sufficient fluorescence to be detected.

Typically, the fluorescence of the moiety involves a combination ofquantum efficiency and lack of photobleaching sufficient that the moietyis detectable above background levels in a single molecule detector,with the consistency necessary for the desired limit of detection,accuracy, and precision of the assay. For example, in some embodiments,the fluorescence of the fluorescent moiety is such that it allowsdetection and/or quantitation of a molecule, e.g., a marker, at a limitof detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001,0.00001, or 0.000001 pg/ml and with a coefficient of variation of lessthan about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% orless, e.g., about 10% or less, in the instruments described herein. Insome embodiments, the fluorescence of the fluorescent moiety is suchthat it allows detection and/or quantitation of a molecule, e.g., amarker, at a limit of detection of less than about 5, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001 pg/ml and with a coefficient of variation of lessthan about 10%, in the instruments described herein.

“Limit of detection,” as that term is used herein, includes the lowestconcentration at which one can identify a sample as containing amolecule of the substance of interest, e.g., the first non-zero value.It can be defined by the variability of zeros and the slope of thestandard curve. For example, the limit of detection of an assay can bedetermined by running a standard curve, determining the standard curvezero value, and adding two standard deviations to that value. Aconcentration of the substance of interest that produces a signal equalto this value is the “lower limit of detection” concentration.

Furthermore, the moiety has properties that are consistent with its usein the assay of choice. In some embodiments, the assay is animmunoassay, where the fluorescent moiety is attached to an antibody;the moiety must not aggregate with other antibodies or proteins, or mustnot undergo any more aggregation than is consistent with the requiredaccuracy and precision of the assay. In some embodiments, fluorescentmoieties that are preferred are fluorescent moieties, e.g., dyemolecules that have a combination of 1) high absorption coefficient; 2)high quantum yield; 3) high photostability (low photobleaching); and 4)compatibility with labeling the molecule of interest (e.g., protein) sothat it can be analyzed using the analyzers and systems of the invention(e.g., does not cause precipitation of the protein of interest, orprecipitation of a protein to which the moiety has been attached).

Fluorescent moieties, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, which are useful in someembodiments of the invention, can be defined in terms of their photonemission characteristics when stimulated by EM radiation. For example,in some embodiments, the invention utilizes a fluorescent moiety, e.g.,a moiety comprising a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 photons whensimulated by a laser emitting light at the excitation wavelength of themoiety, where the laser is focused on a spot of not less than about 5microns in diameter that contains the moiety, and where the total energydirected at the spot by the laser is no more than about 3 microJoules.It will be appreciated that the total energy can be achieved by manydifferent combinations of power output of the laser and length of timeof exposure of the dye moiety. E.g., a laser of a power output of 1 mWcan be used for 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for 0.25 ms,and so on.

In some embodiments, the fluorescent moiety comprises an average of atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, e.g.,fluorescent molecules. In some embodiments, the fluorescent moietycomprises an average of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 fluorescent entities, e.g., fluorescent molecules. In someembodiments, the fluorescent moiety comprises an average of about 1 to11, or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5,or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, orabout 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, orabout 2, 3, 4, 5, 6, or more than about 6 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 2 to 8fluorescent moieties are attached. In some embodiments, the fluorescentmoiety comprises an average of about 2 to 6 fluorescent entities. Insome embodiments, the fluorescent moiety comprises an average of about 2to 4 fluorescent entities. In some embodiments, the fluorescent moietycomprises an average of about 3 to 10 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 3 to 8fluorescent entities. In some embodiments, the fluorescent moietycomprises an average of about 3 to 6 fluorescent entities. By “average”it is meant that, in a given sample that is representative of a group oflabels of the invention, where the sample contains a plurality of thebinding partner-fluorescent moiety units, the molar ratio of theparticular fluorescent entity to the binding partner, as determined bystandard analytical methods, corresponds to the number or range ofnumbers specified. For example, in embodiments wherein the labelcomprises a binding partner that is an antibody and a fluorescent moietythat comprises a plurality of fluorescent dye molecules of a specificabsorbance, a spectrophotometric assay can be used in which a solutionof the label is diluted to an appropriate level and the absorbance at280 nm is taken to determine the molarity of the protein (antibody) andan absorbance at, e.g., 650 nm (for Alexa Fluor 647), is taken todetermine the molarity of the fluorescent dye molecule. The ratio of thelatter molarity to the former represents the average number offluorescent entities (dye molecules) in the fluorescent moiety attachedto each antibody.

1. Dyes

In some embodiments, the invention uses fluorescent moieties thatcomprise fluorescent dye molecules. In some embodiments, the inventionutilizes a fluorescent dye molecule that is capable of emitting anaverage of at least about 50 photons when simulated by a laser emittinglight at the excitation wavelength of the molecule, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the molecule, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye molecule that is capable ofemitting an average of at least about 75 photons when simulated by alaser emitting light at the excitation wavelength of the molecule, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the molecule, and wherein the total energydirected at the spot by the laser is no more than about 3 microJoules.In some embodiments, the invention utilizes a fluorescent dye moleculethat is capable of emitting an average of at least about 100 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe molecule, where the laser is focused on a spot of not less thanabout 5 microns in diameter that contains the molecule, and wherein thetotal energy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye molecule that is capable of emitting an average of at least about150 photons when simulated by a laser emitting light at the excitationwavelength of the molecule, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the molecule, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye molecule that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the molecule, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themolecule, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules.

In some embodiments, the invention uses a fluorescent dye moiety, e.g.,a single fluorescent dye molecule or a plurality of fluorescent dyemolecules, that is capable of emitting an average of at least about 50photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye moiety, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that is capable of emitting anaverage of at least about 100 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye moiety, e.g., a singlefluorescent dye molecule or a plurality of fluorescent dye molecules,that is capable of emitting an average of at least about 150 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, where the laser is focused on a spot of not less than about5 microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye moiety, e.g., a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. In some embodiments, the inventionutilizes a fluorescent dye moiety, e.g., a single fluorescent dyemolecule or a plurality of fluorescent dye molecules, that is capable ofemitting an average of at least about 300 photons when simulated by alaser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. In someembodiments, the invention utilizes a fluorescent dye moiety, e.g., asingle fluorescent dye molecule or a plurality of fluorescent dyemolecules, that is capable of emitting an average of at least about 500photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules.

A non-inclusive list of useful fluorescent entities for use in thefluorescent moieties of the invention is given in Table 2, below. Insome embodiments, the fluorescent dye is selected from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, AlexaFluor 700, Alexa Fluor 750, Fluorescein, B-phycoerythrin,allophycocyanin, PBXL-3, and Qdot 605. In some embodiments, thefluorescent dye is selected from the group consisting of Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 700, Alexa Fluor 750, Fluorescein,B-phycoerythrin, allophycocyanin, PBXL-3, and Qdot 605.

TABLE 2 FLUORESCENT ENTITIES Dye E Ex (nm) E (M)−1 Em (nm) MMw Bimane380 5,700 458 282.31 Dapoxyl 373 22,000 551 362.83 Dimethylaminocoumarin-4-acetic acid 375 22,000 470 344.32 Marina blue 365 19,000 460367.26 8-Anilino naphthalene-1-sulfonic acid 372 480 Cascade blue 37623,000 420 607.42 Alexa Fluor 405 402 35,000 421 1028.26 Cascade blue400 29,000 420 607.42 Cascade yellow 402 24,000 545 563.54 Pacific blue410 46,000 455 339.21 PyMPO 415 26,000 570 582.41 Alexa Fluor 430 43315,000 539 701.75 Atto-425 438 486 NBD 465 22,000 535 391.34 Alexa Fluor488 495 73,000 519 643.41 Fluorescein 494 79,000 518 376.32 Oregon Green488 496 76,000 524 509.38 Atto 495 495 522 Cy2 489 150,000 506 713.78DY-480-XL 500 40,000 630 514.60 DY-485-XL 485 20,000 560 502.59DY-490-XL 486 27,000 532 536.58 DY-500-XL 505 90,000 555 596.68DY-520-XL 520 40,000 664 514.60 Alexa Fluor 532 531 81,000 554 723.77BODIPY 530/550 534 77,000 554 513.31 6-HEX 535 98,000 556 680.07 6-JOE522 75,000 550 602.34 Rhodamine 6G 525 108,000 555 555.59 Atto-520 520542 Cy3B 558 130,000 572 658.00 Alexa Fluor 610 612 138,000 628 AlexaFluor 633 632 159,000 647 ca. 1200 Alexa Fluor 647 650 250,000 668 ca.1250 BODIPY 630/650 625 101,000 640 660.50 Cy5 649 250,000 670 791.99Alexa Fluor 660 663 110,000 690 Alexa Fluor 680 679 184,000 702 AlexaFluor 700 702 192,000 723 Alexa Fluor 750 749 240,000 782B-phycoerythrin 546, 565 2,410,000 575 240,000 R-phycoerythrin 480, 546,565 1,960,000 578 240,000 Allophycocyanin 650 700,000 660 700,000 PBXL-1545 666 PBXL-3 614 662 Atto-tec dyes Name Ex (nm) Em (nm) QY □ (ns) Atto425 436 486 0.9 3.5 Atto 495 495 522 0.45 2.4 Atto 520 520 542 0.9 3.6Atto 560 561 585 0.92 3.4 Atto 590 598 634 0.8 3.7 Atto 610 605 630 0.73.3 Atto 655 665 690 0.3 1.9 Atto 680 680 702 0.3 1.8 Dyomics FluorsMolar absorbance* molecular weight# label Ex (nm) [l · mol−1 · cm−1] Em(nm) [g · mol−1] DY-495/5 495 70,000 520 489.47 DY-495/6 495 70,000 520489.47 DY-495X/5 495 70,000 520 525.95 DY-495X/6 495 70,000 520 525.95DY-505/5 505 85,000 530 485.49 DY-505/6 505 85,000 530 485.49 DY-505X/5505 85,000 530 523.97 DY-505X/6 505 85,000 530 523.97 DY-550 553 122,000578 667.76 DY-555 555 100.000 580 636.18 DY-610 609 81.000 629 667.75DY-615 621 200.000 641 578.73 DY-630 636 200.000 657 634.84 DY-631 637185.000 658 736.88 DY-633 637 180.000 657 751.92 DY-635 647 175.000 671658.86 DY-636 645 190.000 671 760.91 DY-650 653 170.000 674 686.92DY-651 653 160.000 678 888.96 DYQ-660 660 117,000 — 668.86 DYQ-661 661116,000 — 770.90 DY-675 674 110.000 699 706.91 DY-676 674 145.000 699807.95 DY-680 690 125.000 709 634.84 DY-681 691 125.000 708 736.88DY-700 702 96.000 723 668.86 DY-701 706 115.000 731 770.90 DY-730 734185.000 750 660.88 DY-731 736 225.000 759 762.92 DY-750 747 240.000 776712.96 DY-751 751 220.000 779 814.99 DY-776 771 147.000 801 834.98DY-780-OH 770 70.000 810 757.34 DY-780-P 770 70.000 810 957.55 DY-781783 98.000 800 762.92 DY-782 782 102.000 800 660.88 EVOblue-10 651101.440 664 389.88 EVOblue-30 652 102.000 672 447.51 Quantum Dots: Qdot525, QD 565, QD 585, QD 605, QD 655, QD 705, QD 800

Suitable dyes for use in the invention include modified carbocyaninedyes. On such modification comprises modification of an indolium ring ofthe carbocyanine dye to permit a reactive group or conjugated substanceat the number three position. The modification of the indolium ringprovides dye conjugates that are uniformly and substantially morefluorescent on proteins, nucleic acids and other biopolymers, thanconjugates labeled with structurally similar carbocyanine dyes boundthrough the nitrogen atom at the number one position. In addition tohaving more intense fluorescence emission than structurally similar dyesat virtually identical wavelengths, and decreased artifacts in theirabsorption spectra upon conjugation to biopolymers, the modifiedcarbocyanine dyes have greater photostability and higher absorbance(extinction coefficients) at the wavelengths of peak absorbance than thestructurally similar dyes. Thus, the modified carbocyanine dyes resultin greater sensitivity in assays using the modified dyes and theirconjugates. Preferred modified dyes include compounds that have at leastone substituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. Other dye Compounds include compounds thatincorporate an azabenzazolium ring moiety and at least one sulfonatemoiety. The modified carbocyanine dyes that can be used to detectindividual molecules in various embodiments of the invention aredescribed in U.S. Pat. No. 6,977,305, which is herein incorporated byreference in its entirety. Thus, in some embodiments the labels of theinvention utilize a fluorescent dye that includes a substituted indoliumring system in which the substituent on the 3-carbon of the indoliumring contains a chemically reactive group or a conjugated substancegroup.

In some embodiments, the label comprises a fluorescent moiety thatincludes one or more Alexa Fluor dyes (Molecular Probes, Eugene, Oreg.).The Alexa Fluor dyes are disclosed in U.S. Pat. Nos. 6,977,305;6,974,874; 6,130,101; and 6,974,305 which are herein incorporated byreference in their entirety. Some embodiments of the invention utilize adye chosen from the group consisting of Alexa Fluor 647, Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680,Alexa Fluor 700, and Alexa Fluor 750. Some embodiments of the inventionutilize a dye chosen from the group consisting of Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Someembodiments of the invention utilize a dye chosen from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, AlexaFluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Someembodiments of the invention utilize the Alexa Fluor 647 molecule, whichhas an absorption maximum between about 650 and 660 nm and an emissionmaximum between about 660 and 670 nm. The Alexa Fluor 647 dye is usedalone or in combination with other Alexa Fluor dyes.

Currently available organic fluors can be improved by rendering themless hydrophobic by adding hydrophilic groups such as polyethylene.Alternatively, currently sulfonated organic fluors such as the AlexaFluor 647 dye can be rendered less acidic by making them zwitterionic.Particles such as antibodies that are labeled with the modified fluorsare less likely to bind non-specifically to surfaces and proteins inimmunoassays, and thus enable assays that have greater sensitivity andlower backgrounds. Methods for modifying and improving the properties offluorescent dyes for the purpose of increasing the sensitivity of asystem that detects single molecules are known in the art. Preferably,the modification improves the Stokes shift while maintaining a highquantum yield.

2. Quantum Dots

In some embodiments, the fluorescent label moiety that is used to detecta molecule in a sample using the analyzer systems of the invention is aquantum dot. Quantum dots (QDs), also known as semiconductornanocrystals or artificial atoms, are semiconductor crystals thatcontain anywhere between 100 to 1,000 electrons and range from 2-10 nm.Some QDs can be between 10-20 nm in diameter. QDs have high quantumyields, which makes them particularly useful for optical applications.QDs are fluorophores that fluoresce by forming excitons, which aresimilar to the excited state of traditional fluorophores, but have muchlonger lifetimes of up to 200 nanoseconds. This property provides QDswith low photobleaching. The energy level of QDs can be controlled bychanging the size and shape of the QD, and the depth of the QDs'potential. One optical features of small excitonic QDs is coloration,which is determined by the size of the dot. The larger the dot, theredder, or more towards the red end of the spectrum the fluorescence.The smaller the dot, the bluer or more towards the blue end it is. Thebandgap energy that determines the energy and hence the color of thefluoresced light is inversely proportional to the square of the size ofthe QD. Larger QDs have more energy levels which are more closelyspaced, thus allowing the QD to absorb photons containing less energy,i.e., those closer to the red end of the spectrum. Because the emissionfrequency of a dot is dependent on the bandgap, it is possible tocontrol the output wavelength of a dot with extreme precision. In someembodiments the protein that is detected with the single moleculeanalyzer system is labeled with a QD. In some embodiments, the singlemolecule analyzer is used to detect a protein labeled with one QD andusing a filter to allow for the detection of different proteins atdifferent wavelengths.

QDs have broad excitation and narrow emission properties which, whenused with color filtering, require only a single electromagnetic sourceto resolve individual signals during multiplex analysis of multipletargets in a single sample. Thus, in some embodiments, the analyzersystem comprises one continuous wave laser and particles that are eachlabeled with one QD. Colloidally prepared QDs are free floating and canbe attached to a variety of molecules via metal coordinating functionalgroups. These groups include but are not limited to thiol, amine,nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acidsor other ligands. By bonding appropriate molecules to the surface, thequantum dots can be dispersed or dissolved in nearly any solvent orincorporated into a variety of inorganic and organic films. Quantum dots(QDs) can be coupled to streptavidin directly through a maleimide estercoupling reaction or to antibodies through a meleimide-thiol couplingreaction. This yields a material with a biomolecule covalently attachedon the surface, which produces conjugates with high specific activity.In some embodiments, the protein that is detected with the singlemolecule analyzer is labeled with one quantum dot. In some embodiments,the quantum dot is between 10 and 20 nm in diameter. In otherembodiments, the quantum dot is between 2 and 10 nm in diameter. Inother embodiments, the quantum dot is about 2 nm, 3 nm, 4 nm, 5 nm, 6nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 v, 16 nm, 17nm, 18 nm, 19 nm or 20 nm in diameter. Useful Quantum Dots comprise QD605, QD 610, QD 655, and QD 705. A preferred Quantum Dot is QD 605.

C. Binding Partner-Fluorescent Moiety Compositions

The labels of the invention generally contain a binding partner, e.g.,an antibody, bound to a fluorescent moiety to provide the requisitefluorescence for detection and quantitation in the instruments describedherein. Any suitable combination of binding partner and fluorescentmoiety for detection in the single molecule detectors described hereincan be used as a label in the invention. In some embodiments, theinvention provides a label for a marker of a biological state, where thelabel includes an antibody to the marker and a fluorescent moiety. Themarker can be any of the markers described above. The antibody can beany antibody as described above. A fluorescent moiety can be attachedsuch that the label is capable of emitting an average of at least about50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600,700, 800, 900, or 1000 photons when simulated by a laser emitting lightat the excitation wavelength of the moiety, where the laser is focusedon a spot of not less than about 5 microns in diameter that contains thelabel, and wherein the total energy directed at the spot by the laser isno more than about 3 microJoules. In some embodiments, the fluorescentmoiety can be a fluorescent moiety that is capable of emitting anaverage of at least about 50, 100, 150, or 200 photons when simulated bya laser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. Thefluorescent moiety can comprise one or more dye molecules with astructure that includes a substituted indolium ring system wherein thesubstituent on the 3-carbon of the indolium ring contains a chemicallyreactive group or a conjugated substance group. The label compositioncan include a fluorescent moiety that includes one or more dye moleculesselected from the group consisting of Alexa Fluor 488, Alexa Fluor 532,Alexa Fluor 647, Alexa Fluor 700, or Alexa Fluor 750. The labelcomposition can include a fluorescent moiety that includes one or moredye molecules selected from the group consisting of Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 700, or Alexa Fluor 750. The labelcomposition can include a fluorescent moiety that includes one or moredye molecules that are Alexa Fluor 488. The label composition caninclude a fluorescent moiety that includes one or more dye moleculesthat are Alexa Fluor 555. The label composition can include afluorescent moiety that includes one or more dye molecules that areAlexa Fluor 610. The label composition can include a fluorescent moietythat includes one or more dye molecules that are Alexa Fluor 647. Thelabel composition can include a fluorescent moiety that includes one ormore dye molecules that are Alexa Fluor 680. The label composition caninclude a fluorescent moiety that includes one or more dye moleculesthat are Alexa Fluor 700. The label composition can include afluorescent moiety that includes one or more dye molecules that areAlexa Fluor 750.

In some embodiments, the invention provides a composition for thedetection of a marker of a biological state that includes an Alexa Fluormolecule, e.g. an Alexa Fluor molecule selected from the describedgroups, such as an Alexa Fluor 647 molecule attached to an antibodyspecific for the marker. In some embodiments the composition includes anaverage of about 1 to 11, or about 2 to 10, or about 2 to 8, or about 2to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8,or about 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 AlexaFluor 647 molecules attached to an antibody that can detect the marker.In some embodiments the invention provides a composition for thedetection a marker of a biological state that includes an average ofabout 1 to 11, or about 2 to 10, or about 2 to 8, or about 2 to 6, orabout 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to 8, orabout 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8, orabout 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 Alexa Fluor647 molecules attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 2 to 10Alexa Fluor 647 molecules molecule attached to an antibody specific tothe marker. In some embodiments the invention provides a composition forthe detection of a marker of a biological state that includes an averageof about 2 to 8 Alexa Fluor 647 molecules molecule attached to anantibody specific to the marker. In some embodiments the inventionprovides a composition for the detection of a marker of a biologicalstate that includes an average of about 2 to 6 Alexa Fluor 647 moleculesmolecule attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 2 to 4Alexa Fluor 647 molecules molecule attached to an antibody specific tothe marker. In some embodiments the invention provides a composition forthe detection of a marker of a biological state that includes an averageof about 3 to 8 Alexa Fluor 647 molecules molecule attached to anantibody specific to the marker. In some embodiments the inventionprovides a composition for the detection of a marker of a biologicalstate that includes an average of about 3 to 6 Alexa Fluor 647 moleculesmolecule attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 4 to 8Alexa Fluor 647 molecules molecule attached to an antibody specific tothe marker.

Attachment of the fluorescent moiety, or fluorescent entities that makeup the fluorescent moiety, to the binding partner, e.g., an antibody,can be by any suitable means; such methods are well-known in the art andexemplary methods are given in the Examples. In some embodiments, afterattachment of the fluorescent moiety to the binding partner to form alabel for use in the methods of the invention, and prior to the use ofthe label for labeling the marker of interest, it is useful to perform afiltration step. E.g., an antibody-dye label can be filtered prior touse, e.g., through a 0.2 micron filter, or any suitable filter forremoving aggregates. Other reagents for use in the assays of theinvention can also be filtered, e.g., through a 0.2 micron filter, orany suitable filter. Without being bound by theory, it is thought thatsuch filtration removes a portion of the aggregates of the, e.g.,antibody-dye labels. Such aggregates can bind as a unit to the proteinof interest, but, upon release in elution buffer, the aggregates arelikely to disaggregate. Therefore false positives can result whenseveral labels are detected from an aggregate that has bound to only asingle protein molecule of interest. Regardless of theory, filtrationhas been found to reduce false positives in the subsequent assay and toimprove accuracy and precision.

It will be appreciated that immunoassays often employ a sandwich formatin which binding partner pairs, e.g. antibodies, to the same molecule,e.g., a marker, are used. The invention also encompasses binding partnerpairs, e.g., antibodies, wherein both antibodies are specific to thesame molecule, e.g., the same marker, and wherein at least one member ofthe pair is a label as described herein. Thus, for any label thatincludes a binding-partner and a fluorescent moiety, the invention alsoencompasses a pair of binding partners wherein the first bindingpartner, e.g., an antibody, is part of the label, and the second bindingpartner, e.g., an antibody, is, typically, unlabeled and serves as acapture binding partner. In addition, binding partner pairs arefrequently used in FRET assays. FRET assays useful in the invention aredisclosed in U.S. patent application Ser. No. 11/048,660, incorporatedby reference herein in its entirety, and the present invention alsoencompasses binding partner pairs, each of which includes a FRET label

V. Highly Sensitive Analysis of Molecules

In one aspect, the invention provides a method for determining thepresence or absence of a single molecule, e.g., a molecule of a marker,in a sample, by: i) labeling the molecule if present, with a label; andii) detecting the presence or absence of the label, wherein thedetection of the presence of the label indicates the presence of thesingle molecule in the sample. In some embodiments, the method iscapable of detecting the molecule at a limit of detection of less thanabout 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2,1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or0.001 femtomolar. In some embodiments, the method is capable ofdetecting the molecule at a limit of detection of less than about 100femtomolar. In some embodiments, the method is capable of detecting themolecule at a limit of detection of less than about 10 femtomolar. Insome embodiments, the method is capable of detecting the molecule at alimit of detection of less than about 1 femtomolar. In some embodiments,the method is capable of detecting the molecule at a limit of detectionof less than about 0.1 femtomolar. In some embodiments, the method iscapable of detecting the molecule at a limit of detection of less thanabout 0.01 femtomolar. In some embodiments, the method is capable ofdetecting the molecule at a limit of detection of less than about 0.001femtomolar. Detection limits can be determined by use of an appropriatestandard, e.g., National Institute of Standards and Technology referencestandard material.

The methods also provide methods of determining a concentration of amolecule, e.g., a marker indicative of a biological state, in a sampleby detecting single molecules of the molecule in the sample. The“detecting” of a single molecule includes detecting the moleculedirectly or indirectly. In the case of indirect detection, labels thatcorrespond to single molecules, e.g., labels attached to the singlemolecules, can be detected.

In some embodiments, the invention provides a method for determining thepresence or absence of a single molecule of a protein in a biologicalsample, comprising labeling the molecule with a label and detecting thepresence or absence of the label in a single molecule detector, whereinthe label comprises a fluorescent moiety that is capable of emitting atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, wherein the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and wherein the total energy directed at the spot by the laser is nomore than about 3 microJoules. The single molecule detector may, in someembodiments, comprise not more than one interrogation space. The limitof detection of the single molecule in the sample can be less than about10, 1, 0.1, 0.01, or 0.001 femtomolar. In some embodiments, the limit ofdetection is less than about 1 femtomolar. The detecting can comprisedetecting electromagnetic radiation emitted by the fluorescent moiety.The method can further comprise exposing the fluorescent moiety toelectromagnetic radiation, e.g., electromagnetic radiation provided by alaser, such as a laser with a power output of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mW. In someembodiments, the laser stimulus provides light to the interrogationspace for between about 10 to 1000 microseconds, or about 1000, 250,100, 50, 25 or 10 microseconds. In some embodiments, the label furthercomprises a binding partner specific for binding the molecule, such asan antibody. In some embodiments, the fluorescent moiety comprises afluorescent dye molecule, such as a dye molecule that comprises at leastone substituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. In some embodiments, the dye molecule is an AlexaFluor molecule selected from the group consisting of Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. Insome embodiments, the dye molecule is an Alexa Fluor 647 dye molecule.In some embodiments, the fluorescent moiety comprises a plurality ofAlexa Fluor 647 molecules. In some embodiments, the plurality of AlexaFluor 647 molecules comprises about 2 to 4 Alexa Fluor 647 molecules, orabout 3 to 6 Alexa Fluor 647 molecules. In some embodiments, thefluorescent moiety is a quantum dot. The method can further comprisemeasuring the concentration of the protein in the sample.

In some embodiments, detecting the presence or absence of the labelcomprises: (i) directing electromagnetic radiation from anelectromagnetic radiation source to an interrogation space; (ii)providing electromagnetic radiation that is sufficient to stimulate thelabel, such as a fluorescent moiety, to emit photons if the label ispresent in the interrogation space; (iii) translating the interrogationspace through the sample thereby moving the interrogation space todetect the presence or absence of other single molecules; and (iv)detecting photons emitted during the exposure of step (ii). The methodcan further comprise determining a background photon level in theinterrogation space, wherein the background level represents the averagephoton emission of the interrogation space when it is subjected toelectromagnetic radiation in the same manner as in step (ii), butwithout label in the interrogation space. The method can furthercomprise comparing the amount of photons detected in step (iv) to athreshold photon level, wherein the threshold photon level is a functionof the background photon level, wherein an amount of photons detected instep (iv) greater that the threshold level indicates the presence of thelabel, and an amount of photons detected in step (iv) equal to or lessthan the threshold level indicates the absence of the label.

A. Sample

The sample can be any suitable sample. Typically, the sample is abiological sample, e.g., a biological fluid. Such fluids include,without limitation, bronchoalveolar lavage fluid (BAL), blood, serum,plasma, urine, nasal swab, cerebrospinal fluid, pleural fluid, synovialfluid, peritoneal fluid, amniotic fluid, gastric fluid, lymph fluid,interstitial fluid, tissue homogenate, cell extracts, saliva, sputum,stool, physiological secretions, tears, mucus, sweat, milk, semen,seminal fluid, vaginal secretions, fluid from ulcers and other surfaceeruptions, blisters, and abscesses, and extracts of tissues includingbiopsies of normal, malignant, and suspect tissues or any otherconstituents of the body which can contain the target particle ofinterest. Other similar specimens such as cell or tissue culture orculture broth are also of interest.

In some embodiments, the sample is a blood sample. In some embodimentsthe sample is a plasma sample. In some embodiments the sample is a serumsample. In some embodiments, the sample is a urine sample. In someembodiments, the sample is a nasal swab.

B. Sample Preparation

In general, any method of sample preparation can be used that produces alabel corresponding to a molecule of interest, e.g., a marker of abiological state to be measured, where the label is detectable in theinstruments described herein. As is known in the art, sample preparationin which a label is added to one or more molecules can be performed in ahomogeneous or heterogeneous format. In some embodiments, the samplepreparation is formed in a homogenous format. In analyzer systemsemploying a homogenous format, unbound label is not removed from thesample. See, e.g., U.S. patent application Ser. No. 11/048,660. In someembodiments, the particle or particles of interest are labeled byaddition of labeled antibody or antibodies that bind to the particle orparticles of interest.

In some embodiments, a heterogeneous assay format is used, wherein,typically, a step is employed for removing unbound label. Such assayformats are well-known in the art. One particularly useful assay formatis a sandwich assay, e.g., a sandwich immunoassay. In this format, themolecule of interest, e.g., a marker of a biological state, is captured,e.g., on a solid support, using a capture binding partner. Unwantedmolecules and other substances can then optionally be washed away,followed by binding of a label comprising a detection binding partnerand a detectable label, e.g., a fluorescent moiety. Further washesremove unbound label, then the detectable label is released, usuallythough not necessarily still attached to the detection binding partner.In alternative embodiments, sample and label are added to the capturebinding partner without a wash in between, e.g., at the same time. Othervariations will be apparent to one of skill in the art.

In some embodiments, the method for detecting the molecule of interest,e.g., a marker of a biological state, uses a sandwich assay withantibodies, e.g., monoclonal antibodies, as capture binding partners.The method comprises binding molecules in a sample to a capture antibodythat is immobilized on a binding surface, and binding the labelcomprising a detection antibody to the molecule to form a “sandwich”complex. The label comprises the detection antibody and a fluorescentmoiety, as described herein, which is detected, e.g., using the singlemolecule analyzers of the invention. Both the capture and detectionantibodies specifically bind the molecule. Many examples of sandwichimmunoassays are known, and some are described in U.S. Pat. No.4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al.,both of which are incorporated herein by reference. Further examplesspecific to specific markers are described in the Examples.

The capture binding partner can be attached to a solid support, e.g., amicrotiter plate or paramagnetic beads. In some embodiments, theinvention provides a binding partner for a molecule of interest, e.g., amarker of a biological state, attached to a paramagnetic bead. Anysuitable binding partner that is specific for the molecule that it iswished to capture can be used. The binding partner can be an antibody,e.g., a monoclonal antibody. Production and sources of antibodies aredescribed elsewhere herein. It will be appreciated that antibodiesidentified herein as useful as a capture antibody can also be useful asdetection antibodies, and vice versa.

The attachment of the binding partner, e.g., an antibody, to the solidsupport can be covalent or noncovalent. In some embodiments, theattachment is noncovalent. An example of a noncovalent attachmentwell-known in the art is that between biotin-avidin and streptavidin.Thus, in some embodiments, a solid support, e.g., a microtiter plate ora paramagnetic bead, is attached to the capture binding partner, e.g.,an antibody, through noncovalent attachment, e.g.,biotin-avidin/streptavidin interactions. In some embodiments, theattachment is covalent. Thus, in some embodiments, a solid support,e.g., a microtiter plate or a paramagnetic bead, is attached to thecapture binding partner, e.g., an antibody, through covalent attachment.

The capture antibody can be covalently attached in an orientation thatoptimizes the capture of the molecule of interest. For example, in someembodiments, a binding partner, e.g., an antibody, is attached in aorientated manner to a solid support, e.g., a microtiter plate or aparamagnetic microparticle.

An exemplary protocol for oriented attachment of an antibody to a solidsupport is as follows. IgG is dissolved in 0.1 M sodium acetate buffer,pH 5.5 to a final concentration of 1 mg/ml. An equal volume of ice cold20 mM sodium periodate in 0.1 M sodium acetate, pH 5.5 is added. The IgGis allowed to oxidize for 1/2 hour on ice. Excess periodate reagent isquenched by the addition of 0.15 volume of 1 M glycerol. Low molecularweight byproducts of the oxidation reaction are removed byultrafiltration. The oxidized IgG fraction is diluted to a suitableconcentration (typically 0.5 mg/ml IgG) and reacted withhydrazide-activated multiwell plates for at least two hours at roomtemperature. Unbound IgG is removed by washing the multiwell plate withborate buffered saline or another suitable buffer. The plate can bedried for storage if desired. A similar protocol can be followed toattach antibodies to microbeads if the material of the microbead issuitable for such attachment.

In some embodiments, the solid support is a microtiter plate. In someembodiments, the solid support is a paramagnetic bead. An exemplaryparamagnetic bead is Streptavidin C1 (Dynal, 650.01-03). Other suitablebeads will be apparent to those of skill in the art. Methods forattachment of antibodies to paramagnetic beads are well-known in theart. One example is given in Example 2.

The molecule of interest is contacted with the capture binding partner,e.g., capture antibody immobilized on a solid support. Some samplepreparation can be used, e.g., preparation of serum from blood samplesor concentration procedures before the simple is contacted with thecapture antibody. Protocols for binding of proteins in immunoassays arewell-known in the art and are included in the Examples.

The time allowed for binding will vary depending on the conditions; itwill be apparent that shorter binding times are desirable in somesettings, especially in a clinical setting. The use of, e.g.,paramagnetic beads can reduce the time required for binding. In someembodiments, the time allowed for binding of the molecule of interest tothe capture binding partner, e.g., an antibody, is less that about 12,10, 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25,20, 15, 10, or 5 minutes. In some embodiments, the time allowed forbinding of the molecule of interest to the capture binding partner,e.g., an antibody, is less than about 60 minutes. In some embodiments,the time allowed for binding of the molecule of interest to the capturebinding partner, e.g., an antibody, is less than about 40 minutes. Insome embodiments, the time allowed for binding of the molecule ofinterest to the capture binding partner, e.g., an antibody, is less thanabout 30 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the capture binding partner, e.g., anantibody, is less than about 20 minutes. In some embodiments, the timeallowed for binding of the molecule of interest to the capture bindingpartner, e.g., an antibody, is less than about 15 minutes. In someembodiments, the time allowed for binding of the molecule of interest tothe capture binding partner, e.g., an antibody, is less than about 10minutes. In some embodiments, the time allowed for binding of themolecule of interest to the capture binding partner, e.g., an antibody,is less than about 5 minutes.

In some embodiments, following the binding of particles of the moleculeof interest to the capture binding partner, e.g., a capture antibody,particles that bound nonspecifically, as well as other unwantedsubstances in the sample, are washed away leaving substantially onlyspecifically bound particles of the molecule of interest. In otherembodiments, no wash is used between additions of sample and label,which can reduce sample preparation time. Thus, in some embodiments, thetime allowed for both binding of the molecule of interest to the capturebinding partner, e.g., an antibody, and binding of the label to themolecule of interest, is less that about 12, 10, 8, 6, 4, 3, 2, or 1hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., an antibody, andbinding of the label to the molecule of interest, is less that about 60minutes. In some embodiments, the time allowed for both binding of themolecule of interest to the capture binding partner, e.g., an antibody,and binding of the label to the molecule of interest, is less than about40 minutes. In some embodiments, the time allowed for both binding ofthe molecule of interest to the capture binding partner, e.g., anantibody, and binding of the label to the molecule of interest, is lessthan about 30 minutes. In some embodiments, the time allowed for bothbinding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 20 minutes. In some embodiments, the time allowed forboth binding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 15 minutes. In some embodiments, the time allowed forboth binding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 10 minutes. In some embodiments, the time allowed forboth binding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 5 minutes.

Some immunoassay diagnostic reagents, including the capture and signalantibodies used to measure the molecule of interest, can be derived fromanimal sera. Endogenous human heterophilic antibodies, or humananti-animal antibodies, which have the ability to bind toimmunoglobulins of other species, are present in the serum or plasma ofmore than 10% of patients. These circulating heterophilic antibodies caninterfere with immunoassay measurements. In sandwich immunoassays, theseheterophilic antibodies can either bridge the capture and detection(diagnostic) antibodies, thereby producing a false-positive signal, orthey can block the binding of the diagnostic antibodies, therebyproducing a false-negative signal. In competitive immunoassays, theheterophilic antibodies can bind to the analytic antibody and inhibitits binding to the molecule of interest. They can also either block oraugment the separation of the antibody-molecule of interest complex fromfree molecule of interest, especially when antispecies antibodies areused in the separation systems. Therefore, the impact of theseheterophilic antibody interferences is difficult to predict and it canbe advantageous to block the binding of heterophilic antibodies. In someembodiments of the invention, the immunoassay includes the step ofdepleting the sample of heterophilic antibodies using one or moreheterophilic antibody blockers. Methods for removing heterophilicantibodies from samples to be tested in immunoassays are known andinclude: heating the specimen in a sodium acetate buffer, pH 5.0, for 15minutes at 90° C. and centrifuging at 1200 g for 10 minutes;precipitating the heterophilic immunoglobulins using polyethylene glycol(PEG); immunoextracting the interfering heterophilic immunoglobulinsfrom the specimen using protein A or protein G; or adding nonimmunemouse IgG. Embodiments of the methods of the invention contemplatepreparing the sample prior to analysis with the single moleculedetector. The appropriateness of the method of pretreatment can bedetermined. Biochemicals to minimize immunoassay interference caused byheterophilic antibodies are commercially available. For example, aproduct called MAK33, which is an IgG1 monoclonal antibody to h-CK-MM,can be obtained from Boehringer Mannheim. The MAK33 plus productcontains a combination of IgG1 and IgG1-Fab. polyMAK33 contains IgG1-Fabpolymerized with IgG1, and the polyMAC 2b/2a contains IgG2a-Fabpolymerized with IgG2b. Bioreclamation Inc., East Meadow, N.Y., marketsa second commercial source of biochemicals to neutralize heterophilicantibodies known as Immunoglobulin Inhibiting Reagent. This product is apreparation of immunoglobulins (IgG and IgM) from multiple species,mainly murine IgG2a, IgG2b, and IgG3 from Balb/c mice. In someembodiments the heterophilic antibody can be immunoextracted from thesample using methods known in the art, e.g., depleting the sample of theheterophilic antibody by binding the interfering antibody to protein Aor protein G. In some embodiments, the heterophilic antibody can beneutralized using one or more heterophilic antibody blockers.Heterophilic blockers can be selected from the group consisting ofanti-isotype heterophilic antibody blockers, anti-idiotype heterophilicantibody blockers, and anti-anti-idiotype heterophilic antibodyblockers. In some embodiments, a combination of heterophilic antibodyblockers can be used.

Label is added either with or following the addition of sample andwashing. Protocols for binding antibodies and other immunolabels toproteins and other molecules are well-known in the art. If the labelbinding step is separate from that of capture binding, the time allowedfor label binding can be important, e.g., in clinical applications orother time sensitive settings. In some embodiments, the time allowed forbinding of the molecule of interest to the label, e.g., an antibody-dye,is less than about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than about60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 60 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 50 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 40 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 30 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 20 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 15 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 10 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 5 minutes. Excess label is removedby washing.

In some embodiments, the label is not eluted from the protein ofinterest. In other embodiments, the label is eluted from the protein ofinterest. Preferred elution buffers are effective in releasing the labelwithout generating significant background. It is useful if the elutionbuffer is bacteriostatic. Elution buffers used in the invention cancomprise a chaotrope, a buffer, an albumin to coat the surface of themicrotiter plate, and a surfactant selected so as to produce arelatively low background. The chaotrope can comprise urea, aguanidinium compound, or other useful chaotropes. The buffer cancomprise borate buffered saline, or other useful buffers. The proteincarrier can comprise, e.g., an albumin, such as human, bovine, or fishalbumin, an IgG, or other useful carriers. The surfactant can comprisean ionic or nonionic detergent including Tween 20, Triton X-100, sodiumdodecyl sulfate (SDS), and others.

In another embodiment, the solid phase binding assay can be acompetitive binding assay. One such method is as follows. First, acapture antibody immobilized on a binding surface is competitively boundby i) a molecule of interest, e.g., marker of a biological state, in asample, and ii) a labeled analog of the molecule comprising a detectablelabel (the detection reagent). Second, the amount of the label using asingle molecule analyzer is measured. Another such method is as follows.First, an antibody having a detectable label (the detection reagent) iscompetitively bound to i) a molecule of interest, e.g., marker of abiological state in a sample, and ii) an analog of the molecule that isimmobilized on a binding surface (the capture reagent). Second, theamount of the label using a single molecule analyzer is measured. An“analog of a molecule” refers, herein, to a species that competes with amolecule for binding to a capture antibody. Examples of competitiveimmunoassays are disclosed in U.S. Pat. No. 4,235,601 to Deutsch et al.,U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 toBuechler et al., all of which are incorporated herein by reference.

C. Detection of Molecule of Interest and Determination of Concentration

Following elution, the presence or absence of the label in the sample isdetected using a single molecule detector. A sample can contain nolabel, a single label, or a plurality of labels. The number of labelscorresponds to or is proportional to (if dilutions or fractions ofsamples are used) the number of molecules of the molecule of interest,e.g., a marker of a biological state captured during the capture step.

Any suitable single molecule detector capable of detecting the labelused with the molecule of interest can be used. Suitable single moleculedetectors are described herein. Typically the detector is part of asystem that includes an automatic sampler for sampling prepared samples,and, optionally, a recovery system to recover samples.

In some embodiments, the sample is analyzed in a single moleculeanalyzer that uses a laser to illuminate an interrogation spacecontaining a sample, a detector to detect radiation emitted from theinterrogation space, and a scan motor and mirror attached to the motorto translate the interrogation space through the sample. In someembodiments, the single molecule analyzer further comprises a microscopeobjective lens that collects light emitted from the sample as theinterrogation space is translated through the sample, e.g., a highnumerical aperture microscope objective. In some embodiments, the laserand detector are configured in a confocal arrangement. In someembodiments, the laser is a continuous wave laser. In some embodiments,the detector is an avalanche photodiode detector. In some embodiments,the interrogation space is translated through the sample using a mirrorattached to the scan motor. In some embodiments, the interrogation spaceis translated through the sample using multiple mirrors or a prismattached to the scan motor. In some embodiments, the invention providesan analyzer system that includes a sampling system capable ofautomatically sampling a plurality of samples with zero carryoverbetween subsequently measured samples. In some embodiments, theinterrogation space has a volume of more than about 1 μm³, more thanabout 2 μm³, more than about 3 μm³, more than about 4 μm³, more thanabout 5 μm³, more than about 10 μm³, more than about 15 μm³, more thanabout 30 μm³, more than about 50 μm³, more than about 75 μm³, more thanabout 100 μm³, more than about 150 μm³, more than about 200 μm³, morethan about 250 μm³, more than about 300 μm³, more than about 400 μm³,more than about 500 μm³, more than about 550 μm³, more than about 600μm³, more than about 750 μm³, more than about 1000 μm³, more than about2000 μm³, more than about 4000 μm³, more than about 6000 μm³, more thanabout 8000 μm³, more than about 10000 μm³, more than about 12000 μm³,more than about 13000 μm³, more than about 14000 μm³, more than about15000 μm³, more than about 20000 μm³, more than about 30000 μm³, morethan about 40000 μm³, or more than about 50000 μm³′. In someembodiments, the interrogation space is of a volume less than about50000 μm³, less than about 40000 μm³, less than about 30000 μm³, lessthan about 20000 μm³, less than about 15000 μm³, less than about 14000μm³, less than about 13000 μm³, less than about 12000 μm³, less thanabout 11000 μm³, less than about 9500 μm³, less than about 8000 μm³,less than about 6500 μm³, less than about 6000 μm³, less than about 5000μm³, less than about 4000 μm³, less than about 3000 μm³, less than about2500 μm³, less than about 2000 μm³, less than about 1500 μm³, less thanabout 1000 μm³, less than about 800 μm³, less than about 600 μm³, lessthan about 400 μm³, less than about 200 μm³, less than about 100 μm³,less than about 75 pr, less than about 50 μm³, less than about 25 μm³,less than about 20 μm³, less than about 15 μm³, less than about 14 μm³,less than about 13 μm³, less than about 12 μm³, less than about 11 μm³,less than about 10 μm³, less than about 5 μm³, less than about 4 μm³,less than about 3 μm³, less than about 2 μm³, or less than about 1 μm³.In some embodiments, the volume of the interrogation space is betweenabout 1 μm³ and about 10000 μm³. In some embodiments, the interrogationspace is between about 1 μm³ and about 1000 μm³. In some embodiments,the interrogation space is between about 1 μm³ and about 100 μm³. Insome embodiments, the interrogation space is between about 1 μm³ andabout 50 μm³. In some embodiments, the interrogation space is betweenabout 1 μm³ and about 10 μm³. In some embodiments, the interrogationspace is between about 2 μm³ and about 10 μm³. In some embodiments, theinterrogation space is between about 3 μm³ and about 7 μm³.

In some embodiments, the single molecule detector used in the methods ofthe invention uses a sample plate, a continuous wave laser directedtoward a sample plate in which the sample is contained, a high numericalaperture microscope objective lens that collects light emitted from thesample as interrogation space is translated through the sample, whereinthe lens has a numerical aperture of at least about 0.8, an avalanchephotodiode detector to detect radiation emitted from the interrogationspace, and a scan motor with a moveable mirror to translate theinterrogation space through the sample wherein the interrogation spaceis between about 1 μm³ and about 10000 μm³. In some embodiments, thesingle molecule detector used in the methods of the invention uses asample plate, a continuous wave laser directed toward an interrogationspace located within the sample, a high numerical aperture microscopeobjective lens that collects light emitted from the sample as theinterrogation space is translated through the sample, wherein the lenshas a numerical aperture of at least about 0.8, an avalanche photodiodedetector to detect radiation emitted from the interrogation space, and ascan motor for translating the interrogation space through the sample,wherein the interrogation space is between about 1 μm³ and about 1000μm³. In some embodiments, the single molecule detector used in themethods of the invention uses a sample plate, a continuous wave laserdirected toward an interrogation space located within the sample, a highnumerical aperture microscope objective lens that collects light emittedfrom the sample as the interrogation space is translated through thesample, wherein the lens has a numerical aperture of at least about 0.8,an avalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 1 μm³ and about 100 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 1 μm³ and about 10 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample,wherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 2 μm³ and about 10 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 2 μm³ and about 8 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample,wherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 3 μm³ and about 7 μm³. In any of these embodiments, the analyzercan contain only one interrogation space.

In other embodiments, the single molecule detector used in the methodsof the invention uses a sample plate, a continuous wave laser directedtoward a sample plate in which the sample is contained, a high numericalaperture microscope objective lens that collects light emitted from thesample as interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor with a moveable mirror totranslate the interrogation space through the sample wherein theinterrogation space is between about 1 μm³ and about 10000 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 1 μm³ and about 1000 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 1 μm³ and about 100 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 1 μm³ and about 10 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 2 μm³ and about 10 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 2 μm³ and about 8 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 3 μm³ and about 7 μm³. In any ofthese embodiments, the analyzer can contain only one interrogationspace.

In some embodiments, the single molecule detector is capable ofdetermining a concentration for a molecule of interest in a samplewherein the sample can range in concentration over a range of at leastabout 100-fold, 1000-fold, 10,000-fold, 100,000-fold, 300,000-fold,1,000,000-fold, 10,000,000-fold, or 30,000,000-fold.

In some embodiments, the methods of the invention use a single moleculedetector capable detecting a difference of less than about 50%, 40%,30%, 20%, 15%, or 10% in concentration of an analyte between a firstsample and a second sample contained in a sample plate, wherein thevolume of the first sample and the second sample introduced into theanalyzer is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10,5, 4, 3, 2, or 1 μl, and wherein the analyte is present at aconcentration of less than about 100, 90, 80, 70, 60, 50, 40, 30, 20,15, 10, 5, 4, 3, 2, or 1 femtomolar. In some embodiments, the methods ofthe invention use a single molecule detector capable of detecting adifference of less than about 50% in concentration of an analyte betweena first sample and a second sample introduced into the detector, whereinthe volume of the first sample and the second sample introduced into theanalyzer is less than about 100 μl, and wherein the analyte is presentat a concentration of less than about 100 femtomolar. In someembodiments, the methods of the invention use a single molecule detectorcapable detecting a difference of less than about 40% in concentrationof an analyte between a first sample and a second sample that areintroduced into the detector, wherein the volume of the first sample andthe second sample introduced into the analyzer is less than about 50 μl,and wherein the analyte is present at a concentration of less than about50 femtomolar. In some embodiments, the methods of the invention use asingle molecule detector capable detecting a difference of less thanabout 20% in concentration of an analyte between a first sample and asecond sample that are introduced into the detector, wherein the volumeof the first sample and the second sample introduced into the analyzeris less than about 20 μl, and wherein the analyte is present at aconcentration of less than about 20 femtomolar. In some embodiments, themethods of the invention use a single molecule detector capabledetecting a difference of less than about 20% in concentration of ananalyte between a first sample and a second sample that are introducedinto the detector, where the volume of the first sample and the secondsample introduced into the analyzer is less than about 10 μl, andwherein the analyte is present at a concentration of less than about 10femtomolar. In some embodiments, the methods of the invention use asingle molecule detector capable detecting a difference of less thanabout 20% in concentration of an analyte between a first sample and asecond sample that are introduced into the detector, wherein the volumeof the first sample and the second sample introduced into the analyzeris less than about 5 μl, and wherein the analyte is present at aconcentration of less than about 5 femtomolar.

A feature that contributes to the extremely high sensitivity of theinstruments and methods of the invention is the method of detecting andcounting labels, which, in some embodiments, are attached to singlemolecules to be detected or, more typically, correspond to a singlemolecule to be detected. Briefly, the sample contained in the sampleplate is effectively divided into a series of detection events, bytranslating an interrogation space through the sample plate wherein EMradiation from a laser of an appropriate excitation wavelength for thefluorescent moiety used in the label for a predetermined period of timeis directed to the wavelength, and photons emitted during that time aredetected. Each predetermined period of time is a “bin.” If the totalnumber of photons detected in a given bin exceeds a predeterminedthreshold level, a detection event is registered for that bin, i.e., alabel has been detected. If the total number of photons is not at thepredetermined threshold level, no detection event is registered. In someembodiments, the processing sample concentration is dilute enough that,for a large percentage of detection events, the detection eventrepresents only one label passing through the window, which correspondsto a single molecule of interest in the original sample. Accordingly,few detection events represent more than one label in a single bin. Insome embodiments, further refinements are applied to allow greaterconcentrations of label in the processing sample to be detectedaccurately, i.e., concentrations at which the probability of two or morelabels being detected as a single detection event is no longerinsignificant.

Although other bin times can be used without departing from the scope ofthe present invention, in some embodiments the bin times are selected inthe range of about 1 microsecond to about 5 ms. In some embodiments, thebin time is more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750,800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In someembodiments, the bin time is less than about 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600,700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. Insome embodiments, the bin time is about 1 to 1000 microseconds. In someembodiments, the bin time is about 1 to 750 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 250 microseconds. In someembodiments, the bin time is about 1 to 100 microseconds. In someembodiments, the bin time is about 1 to 50 microseconds. In someembodiments, the bin time is about 1 to 40 microseconds. In someembodiments, the bin time is about 1 to 30 microseconds. In someembodiments, the bin time is about 1 to 25 microseconds. In someembodiments, the bin time is about 1 to 20 microseconds. In someembodiments, the bin time is about 1 to 10 microseconds. In someembodiments, the bin time is about 1 to 7.5 microseconds. In someembodiments, the bin time is about 1 to 5 microseconds. In someembodiments, the bin time is about 5 to 500 microseconds. In someembodiments, the bin time is about 5 to 250 microseconds. In someembodiments, the bin time is about 5 to 100 microseconds. In someembodiments, the bin time is about 5 to 50 microseconds. In someembodiments, the bin time is about 5 to 20 microseconds. In someembodiments, the bin time is about 5 to 10 microseconds. In someembodiments, the bin time is about 10 to 500 microseconds. In someembodiments, the bin time is about 10 to 250 microseconds. In someembodiments, the bin time is about 10 to 100 microseconds. In someembodiments, the bin time is about 10 to 50 microseconds. In someembodiments, the bin time is about 10 to 30 microseconds. In someembodiments, the bin time is about 10 to 20 microseconds. In someembodiments, the bin time is about 1 microsecond. In some embodiments,the bin time is about 2 microseconds. In some embodiments, the bin timeis about 3 microseconds. In some embodiments, the bin time is about 4microseconds. In some embodiments, the bin time is about 5 microseconds.In some embodiments, the bin time is about 6 microseconds. In someembodiments, the bin time is about 7 microseconds. In some embodiments,the bin time is about 8 microseconds. In some embodiments, the bin timeis about 9 microseconds. In some embodiments, the bin time is about 10microseconds. In some embodiments, the bin time is about 11microseconds. In some embodiments, the bin time is about 12microseconds. In some embodiments, the bin time is about 13microseconds. In some embodiments, the bin time is about 14microseconds. In some embodiments, the bin time is about 5 microseconds.In some embodiments, the bin time is about 15 microseconds. In someembodiments, the bin time is about 16 microseconds. In some embodiments,the bin time is about 17 microseconds. In some embodiments, the bin timeis about 18 microseconds. In some embodiments, the bin time is about 19microseconds. In some embodiments, the bin time is about 20microseconds. In some embodiments, the bin time is about 25microseconds. In some embodiments, the bin time is about 30microseconds. In some embodiments, the bin time is about 40microseconds. In some embodiments, the bin time is about 50microseconds. In some embodiments, the bin time is about 100microseconds. In some embodiments, the bin time is about 250microseconds. In some embodiments, the bin time is about 500microseconds. In some embodiments, the bin time is about 750microseconds. In some embodiments, the bin time is about 1000microseconds.

In some embodiments, determining the concentration of a particle-labelcomplex in a sample comprises determining the background noise level. Insome embodiments, the background noise level is determined from the meannoise level, or the root-mean-square noise. In other embodiments, atypical noise value or a statistical value is chosen. Often, the noiseis expected to follow a Poisson distribution.

As the interrogation space is translated through the sample, the laserbeam directed to the interrogation space generates a burst of photonswhen a label is encountered. The photons emitted by the label arediscriminated from background light or background noise emission byconsidering only the bursts of photons with energy above a predeterminedthreshold energy level, thereby accounting for the amount of backgroundnoise present in the sample. Background noise typically comprises lowfrequency emission produced, e.g., by the intrinsic fluorescence ofnon-labeled particles that are present in the sample, the buffer ordiluent used in preparing the sample for analysis, Raman scattering andelectronic noise. In some embodiments, the value assigned to thebackground noise is calculated as the average background signal noisedetected in a plurality of bins, which are measurements of photonsignals that are detected in an interrogation space during apredetermined length of time. In some embodiments, background noise iscalculated for each sample as a number specific to that sample.

Given the value for the background noise, a threshold energy level canbe assigned. As discussed above, the threshold value is determined todiscriminate true signals resulting from the fluorescence of a labelfrom the background noise. A threshold value can be chosen such that thenumber of false positive signals from random noise is minimized whilethe number of true signals which are rejected is also minimized. Methodsfor choosing a threshold value include determining a fixed value abovethe noise level and calculating a threshold value based on thedistribution of the noise signal. In one embodiment, the threshold isset at a fixed number of standard deviations above the background level.Assuming a Poisson distribution of the noise, using this method one canestimate the number of false positive signals over the time course ofthe experiment. In some embodiments, the threshold level is calculatedas a value of four standard deviations (σ) above the background noise.For example, given an average background noise level of 200 photons, theanalyzer system establishes a threshold level of 44200 above the averagebackground/noise level of 200 photons to be 256 photons. Thus, in someembodiments, determining the concentration of a label in a sampleincludes establishing the threshold level above which photon signalsrepresent the presence of a label. Conversely, the absence of photonsignals with an energy level greater than the threshold level indicatethe absence of a label.

Many bin measurements are taken to determine the concentration of asample, and the absence or presence of a label is ascertained for eachbin measurement. Typically, 60,000 measurements or more can be made in 1min. 60,000 measurements are made in 1 min when the bin size is 1 ms.For smaller bin sizes the number of measurements is correspondinglylarger, e.g., 6,000,000 measurements per minute equates to a bin size of10 microseconds. Because so many measurements are taken, no singlemeasurement is crucial, thus providing for a high margin of error. Binsthat are determined not to contain a label (“no” bins) are discountedand only the measurements made in the bins that are determined tocontain label (“yes” bins) are accounted in determining theconcentration of the label in the processing sample. Discountingmeasurements made in the “no” bins or bins that are devoid of labelincreases the signal to noise ratio and the accuracy of themeasurements. Thus, in some embodiments, determining the concentrationof a label in a sample comprises detecting the bin measurements thatreflect the presence of a label.

The signal to noise ratio or the sensitivity of the analyzer system canbe increased by minimizing the time that background noise is detectedduring a bin measurement in which a particle-label complex is detected.For example, consider a bin measurement lasting 1 millisecond duringwhich one particle-label complex is detected as it passes across aninterrogation space in 250 microseconds. Under these conditions, 750microseconds of the 1 millisecond are spent detecting background noiseemission. The signal to noise ratio can be improved by decreasing thebin time. In some embodiments, the bin time is 1 millisecond. In otherembodiments, the bin time is 750 microseconds, 500 microseconds, 250microseconds, 100 microseconds, 50 microseconds, 25 microseconds or 10microseconds. Other bin times are as described herein.

Other factors that affect measurements are the brightness or dimness ofthe fluorescent moiety, size of the aperture image or lateral extent ofthe laser beam, the rate at which the interrogation space is translatedthrough the sample, and the power of the laser. Various combinations ofthe relevant factors that allow for detection of label will be apparentto those of skill in the art. In some embodiments, the bin time isadjusted without changing the scan speed. It will be appreciated bythose of skill in the art that as bin time decreases, laser power outputdirected at the interrogation space must increase to maintain a constanttotal energy applied to the interrogation space during the bin time. Forexample, if bin time is decreased from 1000 microseconds to 250microseconds, as a first approximation, laser power output must beincreased approximately four-fold. These settings allow for thedetection of the same number of photons in a 250 microseconds as thenumber of photons counted during the 1000 microseconds given theprevious settings, and allow for faster analysis of sample with lowerbackgrounds and greater sensitivity. In addition, the speed at which theinterrogation space is translated through the sample can be adjusted inorder to speed processing of sample. These numbers are merely exemplary,and the skilled practitioner can adjust the parameters as necessary toachieve the desired result.

In some embodiments, the interrogation space is smaller than the volumeof sample when, for example, the interrogation space is defined by thesize of the spot illuminated by the laser beam. In some embodiments, theinterrogation space can be defined by adjusting the apertures 182 (FIGS.1A & 1B) of the analyzer and reducing the illuminated volume that isimaged by the objective lens to the detector. In embodiments wherein theinterrogation space is defined to be smaller than the cross-sectionalarea of the sample, the concentration of the label can be determined byinterpolation of the signal emitted by the complex from a standard curvethat is generated using one or more samples of known standardconcentrations. In other embodiments, the concentration of the label canbe determined by comparing the measured particles to an internal labelstandard. In embodiments wherein a diluted sample is analyzed, thedilution factor is accounted for when calculating the concentration ofthe molecule of interest in the starting sample.

To determine the concentration of labels in the processing sample, thetotal number of labels contained in the “yes” bins is determinedrelative to the sample volume represented by the total number of bins.Thus, in one embodiment, determining the concentration of a label in aprocessing sample comprises determining the total number of labelsdetected “yes” and relating the total number of detected labels to thetotal sample volume that was analyzed. The total sample volume that isanalyzed is the sample volume through which the interrogation space istranslated in a specified time interval. Alternatively, theconcentration of the label complex in a sample is determined byinterpolation of the signal emitted by the label in a number of binsfrom a standard curve that is generated by determining the signalemitted by labels in the same number of bins by standard samplescontaining known concentrations of the label.

In some embodiments, the number of individual labels detected in a binis related to the relative concentration of the particle in theprocessing sample. At relatively low concentrations, e.g., atconcentrations below about 10⁻¹⁶ M, the number of labels is proportionalto the photon signal detected in a bin. Thus, at low concentrations oflabel the photon signal is provided as a digital signal. At relativelyhigher concentrations, for example at concentrations greater than about10⁻¹⁶ M, the proportionality of photon signal to a label is lost as thelikelihood of two or more labels crossing the interrogation space atabout the same time and being counted as one becomes significant. Thus,in some embodiments, individual particles in a sample of a concentrationgreater than about 10⁻¹⁶ M are resolved by decreasing the length of timeof the bin measurement.

In other embodiments, the total photon signal that is emitted by aplurality of particles that are present in any one bin is detected.These embodiments allow for single molecule detectors of the inventionwherein the dynamic range is at least 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7,7.5, 8, or more than 8 logs.

“Dynamic range,” as that term is used herein, refers to the range ofsample concentrations that can be quantitated by the instrument withoutneed for dilution or other treatment to alter the concentration ofsuccessive samples of differing concentrations, where concentrations aredetermined with accuracy appropriate for the intended use. For example,if a microtiter plate contains a sample of 1 femtomolar concentrationfor an analyte of interest in one well, a sample of 10,000 femtomolarconcentration for an analyte of interest in another well, and a sampleof 100 femtomolar concentration for the analyte in a third well, aninstrument with a dynamic range of at least 4 logs and a lower limit ofquantitation of 1 femtomolar can accurately quantitate the concentrationof all the samples without further treatment to adjust concentration,e.g., dilution. Accuracy can be determined by standard methods, e.g.,measuring a series of standards with concentrations spanning the dynamicrange and constructing a standard curve. Standard measures of fit of theresulting standard curve can be used as a measure of accuracy, e.g., anr² greater than about 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94,0.95, 0.96, 0.97, 0.98, or 0.99.

Dynamic range can be increased by altering how data from the detector isanalyzed, and perhaps using an attenuator between the detector and theinterrogation space. At the low end of the range, the processing sampleis sufficiently dilute that each detection event, i.e., each burst ofphotons above a threshold level in a bin (the “event photons”), likelyrepresents only one label. Under these conditions, the data is analyzedto count detection events as single molecules so that each bin isanalyzed as a simple “yes” or “no” for the presence of label, asdescribed above. For a more concentrated processing sample, where thelikelihood of two or more labels occupying a single bin becomessignificant, the number of event photons in a significant number of binsis substantially greater than the number expected for a single label.For example, the number of event photons in a significant number of binscorresponds to two-fold, three-fold, or more than the number of eventphotons expected for a single label. For these samples, the instrumentchanges its method of data analysis to integrate the total number ofevent photons for the bins of the processing sample. This total isproportional to the total number of labels in all the bins. For an evenmore concentrated processing sample, where many labels are present inmost bins, background noise becomes an insignificant portion of thetotal signal from each bin, and the instrument changes its method ofdata analysis to count total photons per bin (including background). Aneven further increase in dynamic range can be achieved by the use of anattenuator between the sample plate and the detector, whenconcentrations are such that the intensity of light reaching thedetector would otherwise exceed the capacity of the detector foraccurately counting photons, i.e., saturate the detector.

The instrument can include a data analysis system that receives inputfrom the detector and determines the appropriate analysis method for thesample being run, and outputs values based on such analysis. The dataanalysis system can further output instructions to use or not use anattenuator, if an attenuator is included in the instrument.

By utilizing such methods, the dynamic range of the instrument can bedramatically increased. In some embodiments, the instrument is capableof measuring concentrations of samples over a dynamic range of more thanabout 1000 (3 log), 10,000 (4 log), 100,000 (5 log), 350,000 (5.5 log),1,000,000 (6 log), 3,500,000 (6.5 log), 10,000,000 (7 log), 35,000,000(7.5 log), or 100,000,000 (8 log). In some embodiments, the instrumentis capable of measuring concentrations of samples over a dynamic rangeof more than about 100,000 (5 log). In some embodiments, the instrumentis capable of measuring concentrations of samples over a dynamic rangeof more than about 1,000,000 (6 log). In some embodiments, theinstrument is capable of measuring concentrations of samples over adynamic range of more than about 10,000,000 (7 log). In someembodiments, the instrument is capable of measuring the concentrationsof samples over a dynamic range of from about 1 to 10 femtomolar to atleast about 1000, 10,000, 100,000, 350,000, 1,000,000, 3,500,000,10,000,000, or 35,000,000 femtomolar. In some embodiments, theinstrument is capable of measuring the concentrations of samples over adynamic range of from about 1 to 10 femtomolar to at least about 10,000femtomolar. In some embodiments, the instrument is capable of measuringthe concentrations of samples over a dynamic range of from about 1 to 10femtomolar to at least about 100,000 femtomolar. In some embodiments,the instrument is capable of measuring the concentrations of samplesover a dynamic range of from about 1 to 10 femtomolar to at least about1,000,000 femtomolar. In some embodiments, the instrument is capable ofmeasuring the concentrations of samples over a dynamic range of fromabout 1 to 10 femtomolar to at least about 10,000,000.

In some embodiments, an analyzer or analyzer system of the invention iscapable of detecting an analyte, e.g., a biomarker, at a limit ofdetection of less than about 1 nanomolar, or 1 picomolar, or 1femtomolar, or 1 attomolar, or 1 zeptomolar. In some embodiments, theanalyzer or analyzer system is capable of detecting a change inconcentration of the analyte, or of multiple analytes, e.g., a biomarkeror biomarkers, from one sample to another sample of less than about0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 80% when thebiomarker is present at a concentration of less than about 1 nanomolar,or 1 picomolar, or 1 femtomolar, or 1 attomolar, or 1 zeptomolar, in thesamples, and when the size of each of the sample is less than about 100,50, 40, 30, 20, 10, 5, 2, 1, 0.1, 0.01, 0.001, or 0.0001 μl. In someembodiments, the analyzer or analyzer system is capable of detecting achange in concentration of the analyte from a first sample to a secondsample of less than about 20%, when the analyte is present at aconcentration of less than about 1 picomolar, and when the size of eachof the samples is less than about 50 μl. In some embodiments, theanalyzer or analyzer system is capable of detecting a change inconcentration of the analyte from a first sample to a second sample ofless than about 20%, when the analyte is present at a concentration ofless than about 100 femtomolar, and when the size of each of the samplesis less than about 50 μl. In some embodiments, the analyzer or analyzersystem is capable of detecting a change in concentration of the analytefrom a first sample to a second sample of less than about 20%, when theanalyte is present at a concentration of less than about 50 femtomolar,and when the size of each of the samples is less than about 50 μl. Insome embodiments, the analyzer or analyzer system is capable ofdetecting a change in concentration of the analyte from a first sampleto a second sample of less than about 20%, when the analyte is presentat a concentration of less than about 5 femtomolar, and when the size ofeach of the samples is less than about 50 μl. In some embodiments, theanalyzer or analyzer system is capable of detecting a change inconcentration of the analyte from a first sample to a second sample ofless than about 20%, when the analyte is present at a concentration ofless than about 5 femtomolar, and when the size of each of the samplesis less than about 5 μl. In some embodiments, the analyzer or analyzersystem is capable of detecting a change in concentration of the analytefrom a first sample to a second sample of less than about 20%, when theanalyte is present at a concentration of less than about 1 femtomolar,and when the size of each of the samples is less than about 5 μl.

VI. Sample Carryover

Carryover is undesirable in diagnostics. The detection of a molecule ofinterest in one sample cannot compromise the accuracy of the detectionof a molecule of interest in a subsequent sample being tested. Thesingle molecule analyzer described herein is capable of detecting thepresence or absence of a single molecule in one sample followed by thedetection of the presence or absence of a single molecule in asubsequent sample with zero carryover between samples. The inventiondescribed herein provides for an instrument capable of sequentiallydetecting the presence or absence of a single molecule of a particulartype in a first sample, and detecting the presence or absence of asingle molecule of the type in a second sample, wherein the instrumentis adapted and configured so that there is no carryover between thefirst and the second sample. Further provided herein is a method ofsequentially detecting the presence or absence of a single molecule of aparticular type in a first sample, and detecting the presence or absenceof a single molecule of the type in a second sample, wherein there is nocarryover between the first and the second sample.

In some embodiments, multiple samples are run on the same sample plate.In some embodiments, the samples are tested for the same type of singlemolecule of interest. In some embodiments, the type of single moleculetested for in the first sample is not the same type of molecule testedfor in the second sample. This would be the case when running, e.g., apanel where the original sample is divided into multiple samples, eachof which is tested for a different type of single molecule of interest.

In some embodiments, the sample plate contains one sample to be tested.In some embodiments, the sample plate contains two samples to be tested.In some embodiments, multiple samples can be tested on the same sampleplate. In theory, tens, to hundreds, to thousands, or more thanthousands of samples can be run sequentially with zero carryover betweenany two samples tested sequentially. The system is limited to the numberof samples only by the constraints of the sample plate.

Creating a system with zero carryover is simple for systems in which thecontainer or containers for containing the samples being tested aredisposable. In such systems, as long as the detecting means does notcome in contact with the sample, there is no chance of carryover with adisposable container. Disposable containers include items such ascuvettes and capillary tubes. The invention provided herein permits thetesting of sequential samples that are contained within disposable andnon-disposable containers. The invention discloses an instrumentationconfiguration wherein carryover between samples is not possible.

VII. Methods of Use of Single Molecule Analyzer

Further provided herein is a method for detecting the presence orabsence of a single molecule in a sample comprising: (a) directingelectromagnetic radiation from an electromagnetic radiation source to aninterrogation space in the sample; (b) detecting the presence or absenceof a first single molecule in the interrogation space located at a firstposition in the sample; (c) translating the interrogation space throughthe sample to a subsequent position in the sample; (d) detecting thepresence or absence of a subsequent single molecule in the subsequentposition in the sample; and (e) repeating steps (c) and (d) as requiredto detect the presence or absence of a single molecule in more than oneposition of the sample. In some embodiments, the interrogation space hasa volume of more than about 1 μm³, more than about 2 μm³, more thanabout 3 μm³, more than about 4 μm³, more than about 5 μm³, more thanabout 10 μm³, more than about 15 μm³, more than about 30 μm³, more thanabout 50 μm³, more than about 75 μm³, more than about 100 μm³, more thanabout 150 μm³, more than about 200 μm³, more than about 250 μm³, morethan about 300 μm³, more than about 400 μm³, more than about 500 μm³,more than about 550 μm³, more than about 600 μm³, more than about 750μm³, more than about 1000 μm³, more than about 2000 μm³, more than about4000 μm³, more than about 6000 μm³, more than about 8000 μm³, more thanabout 10000 μm³, more than about 12000 μm³, more than about 13000 μm³,more than about 14000 μm³, more than about 15000 μm³, more than about20000 μm³, more than about 30000 μm³, more than about 40000 μm³, or morethan about 50000 μm³. In some embodiments, the interrogation space is ofa volume less than about 50000 μm³, less than about 40000 μm³, less thanabout 30000 μm³, less than about 20000 μm³, less than about 15000 μm³,less than about 14000 μm³, less than about 13000 μm³, less than about12000 μm³, less than about 11000 μm³, less than about 9500 μm³, lessthan about 8000 μm³, less than about 6500 μm³, less than about 6000 μm³,less than about 5000 μm³, less than about 4000 μm³, less than about 3000μm³, less than about 2500 μm³, less than about 2000 μm³, less than about1500 μm³, less than about 1000 μm³, less than about 800 μm³, less thanabout 600 μm³, less than about 400 μm³, less than about 200 μm³, lessthan about 100 μm³, less than about 75 μm³, less than about 50 μm³, lessthan about 25 μm³, less than about 20 μm³, less than about 15 μm³, lessthan about 14 μm³, less than about 13 μm³, less than about 12 μm³, lessthan about 11 μm³, less than about 10 μm³, less than about 5 μm³, lessthan about 4 μm³, less than about 3 μm³, less than about 2 μm³, or lessthan about 1 μm³. In some embodiments, the volume of the interrogationspace is between about 1 μm³ and about 10000 μm³. In some embodimentsthe interrogation space is between about 1 μm³ and about 1000 μm³. Insome embodiments the interrogation space is between about 1 μm³ andabout 100 μm³. In some embodiments the interrogation space is betweenabout 1 μm³ and about 50 μm³. In some embodiments the interrogationspace is between about 1 μm³ and about 10 μm³. In some embodiments, theinterrogation space is between about 2 μm³ and about 10 μm³. In someembodiments, the interrogation space is between about 3 μm³ and about 7μm³.

Further provided herein is a method for detecting the presence orabsence of a single molecule wherein the interrogation space istranslated in a non-linear path. In a further embodiment, the non-linearpath comprises a substantially circular path. In another embodiment, thenon-linear path comprises a helical pattern. The invention provides fora method of detecting the presence or absence of a single molecule in aninterrogation space wherein the interrogation space is translatedthrough the sample. In some embodiments, the method provides for thesample to remain substantially stationary relative to theinstrumentation. In some embodiments, the method provides that thesample is translated with respect to the instrumentation. In someembodiments, both the sample and the electromagnetic radiation aretranslated with respect to one another. In an embodiment where thesample is translated with respect to the instrumentation, the sample canremain stationary within its container, e.g., a microwell. While singlemolecules can diffuse in and out of an interrogation space or a seriesof interrogations spaces, the medium in which the single molecules arepresent remains stationary. Therefore, this system allows for singlemolecule detection without the need for flowing fluid.

EXAMPLES Example 1: Molecule Detection and Standard Curve Generation

FIG. 3 illustrates the detection of single molecules using a device ofthe present invention. The plot shows representative data forfluorescence detected on the vertical axis versus time (msec) on thehorizontal axis. The spikes shown in the graph were generated when thescanning single molecule analyzer encountered one or more labeledmolecules within the interrogation space. The total fluorescent signalcomprises the sum of individual detection events (DE), wherein an eventcomprises fluorescence detected above the background noise. The count ofall the events during the recording can be referred to as the “DEvalue.” At low concentrations, the DE value corresponds to the number ofdetected molecules. At higher concentrations wherein two or moremolecules can pass through the detection spot at once, the number ofmolecules detected can be higher than the DE count.

FIG. 4 illustrates a standard curve generated with a scanning singlemolecule analyzer. To generate the curve, samples were prepared withknown concentrations and measured using a device of the presentinvention. Three curves are shown in the plot. The upper curvecorresponds to the total photons (TP) detected. The middle curvecorresponds to the event photons (EP) detected. The lower curvecorresponds to detected events (DE). The plot shows the values for eachof these measures (“Counts”) on the vertical axis versus the knownsample concentration (pg/ml) on the horizontal axis. The plotted circlesare the counts plotted at their known concentrations. The solid curve isa least squares fit of the data to a four parameter logistics curve. The“+” symbols are the counts plotted at their interpolated concentrationsinstead of their known concentrations. The “+” symbols indicate how wellthe fitted curve passes through the actual data. This data demonstratesthat as the concentration of the sample is varied, there is a clearchange in the number of molecules detected.

Example 2: Sandwich Assays for Biomarkers: Cardiac Troponin I (cTnI)

The Assay: The purpose of this assay is to detect the presence ofcardiac Troponin I (cTNI) in human serum. The assay format comprises atwo-step sandwich immunoassay using a mouse monoclonal capture antibodyand a goat polyclonal detection antibody. Ten microliters of sample arerequired. The working range of the assay is 0-900 pg/ml with a typicalanalytical limit of detection of 1 to 3 pg/mL The assay requires about 4h of bench time to complete.

Materials:

The following materials are used in the procedure described below. Theassay plate comprises a clear 384 well NUNC™ Maxisorp, product 464718.The plate is passively coated overnight at room temperature with amonoclonal antibody comprising BiosPacific A34440228P Lot # A0316 (5μg/ml in 0.05 M sodium carbonate pH 9.6) and blocked with 5% sucrose, 1%BSA in phosphate buffered saline (PBS), and stored at 4° C. For thestandard curve, Human cardiac Troponin I (BiosPacific Cat # J34000352)is used. The diluent for the standard concentrations is human serumimmuno-depleted of endogenous cTNI, aliquoted and stored at −20° C.Standards are diluted in a 96 well, conical, polypropylene plate (NUNC™product #249944). The following buffers and solutions are used: (a)assay buffer (borate buffer saline (BBS) with 1% BSA and 0.1% TritonX-100); (b) passive blocking solution (assay buffer containing 2 mg/mlmouse IgG (Equitech Bio), 2 mg/ml goat IgG (Equitech Bio), and 2 mg/mlMAK33 IgG1 Poly (Roche #11 939 661)); (c) detection antibody (goatpolyclonal antibody affinity purified to Peptide 3 (BiosPacificG-129-C), labeled with fluorescent dye Alexa Fluor 647, and stored at 4°C.); (d) detection antibody diluent (50% assay buffer, 50% passiveblocking solution); (e) wash buffer (borate buffer saline Triton buffer(BBST) (1.0 M borate, 15.0 M sodium chloride, 10% Triton X-100, pH8.3)); (f) elution buffer (BBS with 4M urea, 0.02% Triton X-100 and0.001% BSA); and (g) coupling buffer (0.1 M NaHCO₃).

Preparation of Alexa Fluor 647 Labeled Antibodies:

The detection antibody 0-129-C is prepared by conjugation to Alexa Fluor647. 100 μg of G-129-C is dissolved in 400 μl of the coupling buffer.The antibody solution is concentrated to 50 μl by transferring thesolution into YM-30 filter and subjecting the solution and filter tocentrifugation. The YM-30 filter and antibody are washed three times byadding 400 μl of the coupling buffer. The antibody is recovered byadding 50 μl of coupling buffer to the filter, inverting the filter, andcentrifuging for 1 min at 5,000×g. The resulting antibody solution has aconcentration of about 1-2 μg/μl. Alexa Fluor 647 NHS ester stocksolution is made by reconstituted one vial of Alexa Fluor 647 in 20 μlDMSO. This solution can be stored at −20° C. for up to 1 month. 3 μl ofAlexa Fluor 647 stock solution is mixed with the antibody solution inthe dark for 1 h. Thereafter, 7.5 μl 1 M tris is added to the antibodyAlexa Fluor 647 solution and mixed. The solution is ultrafiltered withYM-30 to remove low molecular weight components. The volume of theretentate, which contains the antibody conjugated to Alexa Fluor 647, isadjusted to 200-400 μl by adding PBS. 3 μl 10% NaN₃ is added to thesolution. The resulting solution is transferred to an Ultrafree 0.22centrifugal unit and centrifuged for 2 min at 12,000×g. The filtratecontaining the conjugated antibody is collected and used in the assays.

Procedure:

Standards are prepared (0-900 pg/ml) by serial dilutions of the stock ofcTnI standard into standard diluent to achieve a range of cTnIconcentrations of between 12 pg/ml-4.3 μg/ml. 10 μl passive blockingsolution and 10 μl of either the standard or a sample are added to eachwell of the appropriate plate. Standards are run in quadruplicate. Theplate is sealed, preferably with a low-fluorescence sealing film,centrifuged for 1 min at 3000 RPM, and incubated for 2 h at 25C withshaking. The plate is washed five times, and centrifuged until the rotorreaches 3000 RPM in an inverted position over a paper towel. A 1 nMworking dilution of detection antibody is prepared, and 20 μl detectionantibody are added to each well. The plate is sealed and centrifuged,and the assay is incubated for 1 h at 25° C. with shaking. 30 μl elutionbuffer are added per well, the plate is sealed and the assay isincubated for % h at 25° C. The plate can be analyzed immediately or canbe stored for up to 48 h at 4° C. prior to analysis.

For analysis, 20 μl per well are acquired at 40 μl/minute, and 5 μl areanalyzed at a 16.7 mm/sec scan rate. The data is analyzed based on athreshold of 4 standard deviations (o). The raw signal is plotted versusconcentration of the standards. A linear fit is performed for the lowconcentration range, and a non-linear fit is performed for the fullstandard curve. The limit of detection (LOD) is calculated as LOD=(3×σof zero samples)/slope of linear fit. The concentrations of the samplesare determined from the linear or non-linear equation appropriate forthe sample signal.

The sample plate is then loaded into the scanning single moleculeanalyzer. Individually-labeled antibodies are measured by translatingthe interrogation space through the sample at a speed such that theemission from only one fluorescent label is detected in a defined spacefollowing laser excitation. The total fluorescent signal is a sum of theindividual detection events as described above.

Example 3: Sandwich Bead-Based Assays for TnI

The assays described above uses a microtiter plate format where theplastic surface is used to immobilize target molecules. The singleparticle analyzer system is also compatible with assays performed insolution using microparticles or beads to separate bound and unboundentities.

Materials:

MyOne Streptavidin C1 microparticles (MPs) are obtained from Dynal(650.01-03, 10 mg/ml stock). Buffers used include: (a) 10× borate buffersaline Triton Buffer (BBST) (1.0 M borate, 15.0 M sodium chloride, 10%Triton X-100, pH 8.3); (b) assay buffer (2 mg/ml normal goat IgG, 2mg/ml normal mouse IgG, and 0.2 mg/ml MAB-33-IgG-Polymer in 0.1 M Tris(pH 8.1), 0.025 M EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100, and0.1% NaN₃, stored at 4° C.); and (c) elution buffer (BBS with 4 M urea,0.02% Triton X-100, and 0.001% BSA, stored at 2-8° C.). Antibodies usedin the sandwich bead-based assay include: (a) Bio-Ab (A34650228P(BiosPacific) with 1-2 biotins per IgG); and (b) Det-Ab (G-129-C(BiosPacific) conjugated to Alexa Fluor 647, 2-4 fluors per IgG). Thestandard is recombinant human cardiac troponin I (BiosPacific, cat #J34120352). The calibrator diluent is 30 mg/ml BSA in tris bufferedsaline (TBS) with EDTA.

Microparticles Coating:

100 μl of the MPs stock solution is placed in an Eppendorf tube. The MPsare washed three times with 100 μl BBST wash buffer by applying amagnet, removing the supernatant, removing the magnet, and resuspendingin wash buffer. After washing, the MPs are resuspended in 100 μl ofassay buffer and 15 μg of Bio-Ab are added. The mixture is incubated for1 h at room temperature with constant mixing. The MPs are washed fivetimes with 1 ml wash buffer as described above. After the washes the MPsare resuspended in 15 ml of assay buffer (or 100 μl to store at 4° C.).

Preparation of Standard and Samples:

The standard is diluted with calibrator diluent to prepare a properstandard curve, typically ranging from 200 pg/ml to 0.1 pg/ml. Frozenserum and plasma samples are centrifuged 10 min at room temperature at13,000 rpm. Clarified serum or plasma is removed carefully to avoidpellets or floaters and transferred to fresh tubes. 50 μl of eachstandard or sample is pipetted into appropriate wells.

Capture Target:

After resuspension to 15 ml in assay buffer comprising 400 mM NaCl, 150μl of the MPs are added to each well. The mixture is incubated on aBoekel Jitterbug Microplate Incubator Shaker at room temperature for 1h.

Washes and Detection:

The plate is placed on a magnet and the supernatant is removed afterallowing the magnets to capture the MPs. After removing the plate fromthe magnet, 250 μl of wash buffer are added. Again, the plate is placedon a magnet and the supernatant is removed after allowing the magnets tocapture the MPs. 20 μl Det-Ab are added per well. If necessary, Det-Abto 500 ng/ml is first diluted in assay buffer comprising 400 mM NaCl.The mixture is incubated on a Boekel Jitterbug Microplate IncubatorShaker at room temperature for 30 min. The plate is washed as describedthree times with wash buffer. After washing, 250 μl of wash buffer areadded and the samples are transferred into a new 96-well plate. The washstep is repeated twice. 20 μl of elution buffer are then added and themixture is incubated on Boekel Jitterbug Microplate Incubator Shaker atroom temperature for 30 min.

Filter MPs and Transfer to 384-Well Plate:

The standard and samples are transferred into a 384-well filter plateplaced on top of a 384-well assay plate. The plate is centrifuged atroom temperature at 3000 rpm. The filter plate is removed and theappropriate calibrators are added. The plate is covered and is ready forscanning single molecule detector.

Scanning Single Molecule Detector:

A sample in a sample well is scanned using an electromagnetic radiationsource. The interrogation space is translated through the sample. Thesample is scanned at a speed that is sufficiently slow so thatindividually-labeled antibodies are measured during the sample scan.This is achieved by setting the interrogation space such that theemission of only one fluorescent molecule, if present, is detected in adefined space following laser excitation. With each signal representinga digital event, this configuration enables extremely high analyticalsensitivities. Total fluorescent signal is determined as a sum of theindividual digital events. Each molecule counted is a positive datapoint with hundreds to thousands of detected events/sample. The limit ofdetection the cTnI assay of the invention is determined by the mean plus3 a method (see above).

Although preferred embodiments of the present invention have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein can be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-40. (canceled)
 41. An analyzer, comprising: (a) an optical element fordirecting electromagnetic radiation from an electromagnetic radiationsource to a moveable interrogation space in a microwell of a microtiterplate for confining a processing sample; (b) a detector for detectingelectromagnetic radiation emitted from a photon emitting moiety in themoveable interrogation space when the moiety is present in theprocessing sample; and (c) a processor operatively connected to thedetector, wherein the processor is configured to: determine the presenceof the photon emitting moiety in the moveable interrogation space ineach of a plurality of bins by identifying bins having a photon valuegreater than a threshold photon value, wherein each bin having a photonvalue greater than the threshold photon value represents an individualdetection event, and determine the amount of the photon emitting moietyin the processing sample as a function of a sum the individual detectionevents.
 42. The analyzer of claim 41, wherein an electromagneticradiation beam from the electromagnetic radiation source moves relativeto the microwell.
 43. The analyzer of claim 41, wherein the microwellmoves relative to a fixed electromagnetic beam from the electromagneticradiation source.
 44. The analyzer of claim 41, wherein the microwelland an electromagnetic radiation beam from the electromagnetic radiationsource move relative to each other.
 45. The analyzer of claim 41,wherein the processor is further configured to determine the thresholdphoton value.
 46. The analyzer of claim 45, wherein the threshold photonvalue corresponds to a background photon value in the processing sample.47. The analyzer of claim 45, wherein the threshold photon value is afunction of the background photon value in the processing sample. 48.The analyzer of claim 47, wherein the threshold photon value is a fixednumber of standard deviations above the background photon value.
 49. Theanalyzer of claim 46, wherein the processor is further configured todetermine the background photon value from the value of the photonemission from the moveable interrogation space at a plurality ofpositions in the processing sample.
 50. The analyzer of claim 46,wherein the processor is further configured to determine the backgroundphoton value from the value of the photon emission from the moveableinterrogation space at a plurality of positions in the processing samplewhere the photon emitting moiety is absent.
 51. The analyzer of claim46, wherein the processor is further configured to determine thebackground photon value as an average photon emission from the moveableinterrogation space at a plurality of positions in the processing samplewhere the photon emitting moiety is absent.
 52. The analyzer of claim46, wherein the processor is further configured to determine thebackground photon value as a mean photon emission level or aroot-mean-square photon emission level in the moveable interrogationspace.
 53. The analyzer of claim 46, wherein the background photon valueis calculated for each processing sample as a number specific to theprocessing sample.
 54. The analyzer of claim 41, wherein the opticalelement comprises a microscope objective, and wherein a depth of fieldof the microscope objective and a diameter of an aperture imaged by themicroscope objective together define the moveable interrogation space.55. The analyzer of claim 41, wherein the optical element comprises amicroscope objective, and wherein a depth of field of the microscopeobjective and a lateral extent of an electromagnetic radiation beamtogether define the moveable interrogation space.
 56. The analyzer ofclaim 41, further comprising an attenuator operatively connected betweenthe moveable interrogation space and the detector, wherein theattenuator is configured to (i) receive electromagnetic radiationemitted from the moveable interrogation space and (ii) attenuate theelectromagnetic radiation from the moveable interrogation space when anumber of photons detected in one or more bins exceeds a saturationthreshold.
 57. The analyzer of claim 41, wherein the processor isfurther configured to determine the concentration of the photon emittingmoiety by measuring a total number of photons per bin.
 58. The analyzerof claim 41, wherein the total energy received by the interrogationspace from the electromagnetic radiation source during each bin is 0.1to 10 micoJoules.
 59. An analyzer, comprising: (a) an optical elementfor directing electromagnetic radiation from an electromagneticradiation source to a moveable interrogation space in a microwell of amicrotiter plate for confining a processing sample; (b) a detector fordetecting electromagnetic radiation emitted from a photon emittingmoiety in the moveable interrogation space when the moiety is present inthe processing sample; and (c) a processor operatively connected to thedetector, wherein the processor is configured to: determine the presenceof the photon emitting moiety in the moveable interrogation space ineach of a plurality of bins by identifying bins having a photon valuegreater than a threshold value, and compare the number of bins having aphoton value greater than the threshold value to a standard curve.
 60. Amethod, comprising: (a) directing electromagnetic radiation from anelectromagnetic radiation source to an interrogation space in theprocessing sample contained in a microwell of a microtiter plate; (b)moving the interrogation space through the processing sample to aplurality of positions in the processing sample; (c) detectingelectromagnetic radiation emitted from a photon emitting moiety in theinterrogation space when the moiety is present at the plurality ofpositions; (d) determining the presence of the photon emitting moiety inthe moveable interrogation space in each of a plurality of bins byidentifying bins having a photon value greater than a threshold photonvalue, wherein each bin having a photon value greater than the thresholdphoton value represents an individual detection event; and (e)determining the amount of the photon emitting moiety in the processingsample as a function of a sum of individual detection events.